U.S. patent application number 17/047947 was filed with the patent office on 2021-06-10 for novel rsv rna molecules and compositions for vaccination.
This patent application is currently assigned to CureVac AG. The applicant listed for this patent is CureVac AG. Invention is credited to Regina HEIDENREICH, Johannes LUTZ, Benjamin PETSCH, Susanne RAUCH.
Application Number | 20210170017 17/047947 |
Document ID | / |
Family ID | 1000005458628 |
Filed Date | 2021-06-10 |
United States Patent
Application |
20210170017 |
Kind Code |
A1 |
LUTZ; Johannes ; et
al. |
June 10, 2021 |
NOVEL RSV RNA MOLECULES AND COMPOSITIONS FOR VACCINATION
Abstract
The present invention is directed to an artificial nucleic acid,
particularly to an artificial RNA suitable for use in treatment
and/or prophylaxis of an infection with Respiratory syncytial virus
(RSV) or a disorder related to such an infection. The invention
further concerns a method of treating or preventing a disorder or a
disease, first and second medical uses of the artificial RNA,
compositions, and vaccines. Further, the invention is directed to a
kit, particularly to a kit of parts, comprising the artificial RNA,
compositions and vaccines.
Inventors: |
LUTZ; Johannes; (Tubingen,
DE) ; RAUCH; Susanne; (Tubingen, DE) ;
HEIDENREICH; Regina; (Tubingen, DE) ; PETSCH;
Benjamin; (Tubingen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CureVac AG |
Tubingen |
|
DE |
|
|
Assignee: |
CureVac AG
Tubingen
DE
|
Family ID: |
1000005458628 |
Appl. No.: |
17/047947 |
Filed: |
April 17, 2019 |
PCT Filed: |
April 17, 2019 |
PCT NO: |
PCT/EP2019/060000 |
371 Date: |
October 15, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/04 20180101;
C12N 2760/18534 20130101; C12N 15/88 20130101; A61P 31/14 20180101;
A61K 39/155 20130101; A61K 2039/53 20130101; C12N 2760/18522
20130101; C07K 14/005 20130101; C12N 7/00 20130101; A61K 2039/6018
20130101 |
International
Class: |
A61K 39/155 20060101
A61K039/155; C07K 14/005 20060101 C07K014/005; C12N 7/00 20060101
C12N007/00; A61P 31/14 20060101 A61P031/14; A61P 37/04 20060101
A61P037/04; C12N 15/88 20060101 C12N015/88 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 2018 |
EP |
PCT/EP2018/059799 |
Apr 26, 2018 |
EP |
PCT/EP2018/060810 |
May 3, 2018 |
EP |
PCT/EP2018/061423 |
Claims
1. An artificial RNA comprising a) at least one heterologous 5'
untranslated region (5'-UTR) and/or at least one heterologous 3'
untranslated region (3'-UTR); and b) at least one coding sequence
operably linked to said 3'-UTR and/or 5'-UTR encoding at least one
antigenic peptide or protein derived from a RSV fusion (F) protein
or a fragment or variant thereof.
2. Artificial RNA according to claim 1, wherein the at least one
heterologous 3'-UTR comprises a nucleic acid sequence derived from
a 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1,
COX6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or a
variant of any one of these genes.
3. Artificial RNA according to claim 1 or 2, wherein the at least
one heterologous 5'-UTR comprises a nucleic acid sequence derived
from a 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1,
ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or
from a homolog, a fragment or variant of any one of these
genes.
4. Artificial RNA according to any one of the preceding claims,
comprising a-1. at least one 5'-UTR derived from a 5'-UTR of a
HSD174 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a PSMB3 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or a-2. at least one 5'-UTR
derived from a 5'-UTR of a NDUFA4 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a PSMB3 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or a-3. at least one 5'-UTR derived from a 5'-UTR of a SLC7A3 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a PSMB3
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or a-4. at least one 5'-UTR from a 5'-UTR of a
NOSIP gene, or from a corresponding RNA sequence, homolog, fragment
or variant thereof and at least one 3'-UTR derived from a 3'-UTR of
a PSMB3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof; or a-5. at least one 5'-UTR derived
from a 5'-UTR of a MP68 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof and at least one 3'-UTR
derived from a 3'-UTR of a PSMB3 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof; or b-1. at least
one 5'-UTR derived from a 5'-UTR of a UBQLN2 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a RPS9 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or b-2. at least one 5'-UTR derived from a 5'-UTR of a
ASAH1 gene, or from a corresponding RNA sequence, homolog, fragment
or variant thereof and at least one 3'-UTR derived from a 3'-UTR of
a RPS9 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof; or b-3. at least one 5'-UTR derived
from a 5'-UTR of a HSD17B4 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a RPS9 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or b-4. at least one 5'-UTR derived from a 5'-UTR of a HSD17B4
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof and at least one 3'-UTR derived from a 3-UTR of a
CASP1 gene, or from a corresponding RNA sequence, homolog, fragment
or variant thereof; or b-5. at least one 5'-UTR derived from a
5'-UTR of a NOSIP gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof and at least one 3T-UTR
derived from a 3'-UTR of a COX6B1 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof; or c-1. at least
one 5'-UTR derived from a 5'-UTR of a NDUFA4 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a RPS9 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or c-2. at least one 5'-UTR derived from a 5'-UTR of a
NOSIP gene, or from a corresponding RNA sequence, homolog, fragment
or variant thereof and at least one 3'-UTR derived from a 3'-UTR of
a NDUFA1 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof; or c-3. at least one 5'-UTR derived
from a 5'-UTR of a NDUFA4 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a COX6B1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or c-4. at least one 5'-UTR derived from a 5'-UTR of a NDUFA4 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a NDUFA1
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or c-5. at least one 5'-UTR derived from a 5'-UTR
of a ATP5A1 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a PSMB3 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or d-1. at least one 5'-UTR
derived from a 5'-UTR of a RPL31 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a PSMB3 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or d-2. at least one 5'-UTR derived from a 5'-UTR of a ATP5A1 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a CASP1
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or d-3. at least one 5'-UTR derived from a 5'-UTR
of a SLC7A3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a GNAS gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or d-4. at least one 5'-UTR
derived from a 5'-UTR of a HSD17B4 gene, or from a corresponding
RNA sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a NDUFA1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or d-5. at least one 5'-UTR derived from a 5'-UTR of a SLC7A3 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a NDUFA1
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or e-1. at least one 5'-UTR derived from a 5'-UTR
of a TUBB4B gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a RPS9 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or e-2. at least one 5'-UTR
derived from a 5'-UTR of a RPL31 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a RPS9 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or e-3. at least one 5'-UTR derived from a 5'-UTR of a MP68 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a RPS9
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or e-4. at least one 5'-UTR derived from a 5'-UTR
of a NOSIP gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a RPS9 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or e-5. at least one 5'-UTR
derived from a 5'-UTR of a ATP5A1 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a RPS9 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or e-6. at least one 5'-UTR derived from a 5'-UTR of a ATP5A1 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a COX6B1
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or f-1. at least one 5'-UTR derived from a 5'-UTR
of a ATP5A1 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a GNAS gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or f-2. at least one 5'-UTR
derived from a 5'-UTR of a ATP5A1 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a NDUFA1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or f-3. at least one 5'-UTR derived from a 5'-UTR of a HSD17B4
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof and at least one 3'-UTR derived from a 3-UTR of a
COX6B1 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof; or f-4. at least one 5'-UTR derived
from a 5'-UTR of a HSD17B4 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3-UTR derived from a 3'-UTR of a GNAS gene, or from a corresponding
RNA sequence, homolog, fragment or variant thereof; or f-5. at
least one 5'-UTR derived from a 5'-UTR of a MP68 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a COX6B1 ene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or g-1. at least one 5'-UTR derived from a 5'-UTR of a
MP68 gene, or from a corresponding RNA sequence, homolog, fragment
or variant thereof and at least one 3-UTR derived from a 3'-UTR of
a NDUFA1 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof; or g-2. at least one 5'-UTR derived
from a 5'-UTR of a NDUFA4 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a CASP1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or g-3. at least one 5'-UTR derived from a 5'-UTR of a NDUFA4 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a GNAS
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or g-4. at least one 5'-UTR derived from a 5'-UTR
of a NOSIP gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a CASP1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or g-5. at least one 5'-UTR
derived from a 5'-UTR of a RPL31 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a CASP1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or h-1. at least one 5'-UTR derived from a 5'-UTR of a RPL31 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a COX6B1
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or h-2. at least one 5'-UTR derived from a 5'-UTR
of a RPL31 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a GNAS gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or h-3. at least one 5'-UTR
derived from a 5'-UTR of a RPL31 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a NDUFA1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or h-4. at least one 5'-UTR derived from a 5'-UTR of a SLC7A3 gene,
or from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a CASP1
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof; or h-5. at least one 5'-UTR derived from a 5'-UTR
of a SLC7A3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a COX6B1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or i-1. at least one 5'-UTR
derived from a 5'-UTR of a SLC7A3 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a RPS9 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof.
i-2, at least one 5'-UTR derived from a 5'-UTR of a RPL32 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof and at least one 3'-UTR derived from a 3'-UTR of a ALB7
gene, or from a corresponding RNA sequence, homolog, fragment or
variant thereof. i-3. at least one 3'-UTR derived from a 3'-UTR of
a alpha-globin gene gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof.
5. Artificial RNA according to claim 4 comprising UTR elements
according to a-1 (HSD17B4/PSMB3), a-4 (NDUFA4/PSMB3), c-1
(NDUFA4/RPS9), e-4 (NOSIP/RPS9), g-2 (NDUFA4/CASP1), i-2
(RPL32/ALB7), or i-3 (alpha-globin), preferably a-1
(HSD17B4/PSMB3)
6. Artificial RNA according to any one of the preceding claims,
wherein said 5'-UTR derived from a HSD17B4 gene comprises or
consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or 2 or a fragment
or a variant thereof; said 5'-UTR derived from a ASAH1 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or 4 or a
fragment or a variant thereof; said 5'-UTR derived from a ATP5A1
gene comprises or consists of a nucleic acid sequence being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5
or 6 or a fragment or a variant thereof; said 5'-UTR derived from a
MP68 gene comprises or consists of a nucleic acid sequence being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7
or 8 or a fragment or a variant thereof; said 5'-UTR derived from a
NDUFA4 gene comprises or consists of a nucleic acid sequence being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9
or 10 or a fragment or a variant thereof; said 5'-UTR derived from
a NOSIP gene comprises or consists of a nucleic acid sequence being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
11 or 12 or a fragment or a variant thereof; said 5'-UTR derived
from a RPL31 gene comprises or consists of a nucleic acid sequence
being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO: 13 or 14 or a fragment or a variant thereof; said 5'-UTR
derived from a RPL32 gene comprises or consists of a nucleic acid
sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO: 21 or 22 or a fragment or a variant thereof; said
5'-UTR derived from a SLC7A3 gene comprises or consists of a
nucleic acid sequence being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NO: 15 or 16 or a fragment or a variant
thereof; said 5'-UTR derived from a TUBB48 gene comprises or
consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 or 18 or a
fragment or a variant thereof; said 5'-UTR derived from a UBQLN2
gene comprises or consists of a nucleic acid sequence being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
19 or 20 or a fragment or a variant thereof; said 3'-UTR derived
from a PSMB3 gene comprises or consists of a nucleic acid sequence
being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID
NO: 23 or 24 or a fragment or a variant thereof; said 3'-UTR
derived from a CASP1 gene comprises or consists of a nucleic acid
sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO: 25 or 26 or a fragment or a variant thereof; said
3'-UTR derived from a COX6B1 gene comprises or consists of a
nucleic acid sequence being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NO: 27 or 28 or a fragment or a variant
thereof; said 3'-UTR derived from a GNAS gene comprises or consists
of a nucleic acid sequence being identical or at least 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NO: 29 or 30 or a fragment or a
variant thereof; said 3'-UTR derived from a NDUFA1 gene comprises
or consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 31 or 32 or a
fragment or a variant thereof; said 3'-UTR derived from a RPS9 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 or 34 or
a fragment or a variant thereof; 1 said 3'-UTR derived from a ALB7
gene comprises or consists of a nucleic acid sequence being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
35 or 36 or a fragment or a variant thereof; said 3'-UTR derived
from a alpha-globin gene comprises or consists of a nucleic acid
sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO: 37 or 38 or a fragment or a variant thereof.
7. Artificial RNA according to any one of the preceding claims,
wherein the at least one antigenic peptide or protein derived from
RSV F protein is a full-length F protein (F0) or an F protein with
deleted C-terminus (F-del), or a fragment or a variant thereof.
8. Artificial RNA according to any one of the preceding claims,
wherein the RSV F protein is designed to stabilize the antigen in
pre-fusion conformation.
9. Artificial RNA according to any one of the preceding claims,
wherein the RSV F protein comprises a DSCav1 mutation (S155C.
S290C, S190F, and V207L), or a fragment or a variant thereof.
10. Artificial RNA according to any one of the preceding claims,
wherein the RSV F protein comprises the two subunits F2 and F1 in a
single polypeptide chain, wherein F2 and F1 are connected via a
linker element, preferably a GS linker, to generate a stable
F2-linker-F1 protein, wherein said F2-linker-F1 protein preferably
lacks aa104-aa144.
11. Artificial RNA according to any one of the preceding claims,
wherein the RSV F protein comprises at least one further mutation
selected from (S46G, A149C, S215P, Y458C, K465Q), (S46G, E92D,
A149C, S215P, Y458C, K465Q), (S46G, N671, E92D, A149C, S215P,
Y458C, K465Q), (A149C, Y458C), (N183GC, N428C), (Q98C, Q361C, S46G,
E92D, L95M, S215P, I217P, I221M, R429K, K465Q), (Q98C, Q361C, L95M,
I221M, R429K), or (N183GC, N428C, S46G, N671, E92D, S215P, K465Q)
or a fragment or a variant thereof.
12. Artificial RNA according to any one of the preceding claims,
wherein the RSV F protein is selected from F0, F-del, F0_DSCav1,
F_DSCav1_mut0, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3,
F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6, F_DSCav1_mut7,
F_DSCav1_mut8, F-del_DSCav1, F-del_DSCav1_mut0, F-del_DSCav1_mut1,
F-del_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut4,
F-del_DSCav1_mut5, F-del_DSCav1_mut6, F-del_DSCav1_mut7,
F-del_DSCav1_mut8 or a fragment or a variant thereof.
13. Artificial RNA according to any one of the preceding claims,
wherein the at least one coding sequence encodes at least one of
the amino acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636,
2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695,
6064, 6433, 6802, 7171, 7540, 7909, 8279-9683, 11726, 12095, 12464,
12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571,
16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106,
19475 or a fragment or variant of any of these sequences.
14. Artificial RNA according to any one of the preceding claims,
wherein the at least one coding sequence comprises at least one of
the nucleic acid sequences being identical or at least 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NOs: 69-77, 484-492, 899-906,
1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751, 3113-3120,
3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965, 5327-5334,
5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179, 7541-7548,
7910-7917, 11727-11734, 12096-12103, 12465-12472, 12834-12841,
13941-13948, 14310-14317, 14679-14686, 15048-15055, 15417-15424,
15786-15793, 13203-13210, 13572-13579, 16155-16162, 16524-16531,
16893-16900, 17262-17269, 17631-17638, 18000-18007, 18369-18376,
18738-18745, 19107-19114, 19476-19483, 21363-21384, 21389-21410 or
a fragment or a fragment or variant of any of these sequences.
15. Artificial RNA according to any one of the preceding claims,
wherein the at least one coding sequence is a codon modified coding
sequence, wherein the amino acid sequence encoded by the at least
one codon modified coding sequence is preferably not being modified
compared to the amino acid sequence encoded by the corresponding
wild type coding sequence.
16. Artificial RNA according to claim 15, wherein the at least one
codon modified coding sequence is selected from C maximized coding
sequence, CAI maximized coding sequence, human codon usage adapted
coding sequence, G/C content modified coding sequence, and G/C
optimized coding sequence, or any combination thereof.
17. Artificial RNA according to claim 15 or 16, wherein the at
least one coding sequence comprises a codon modified coding
sequence comprising a nucleic acid sequence being identical or at
least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 70-77,
485-492, 899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382,
2744-2751, 3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596,
4958-4965, 5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810,
7172-7179, 7541-7548, 7910-7917, 11728-11734, 12097-12103,
12465-12472, 12834-12841, 13941-13948, 14310-14317, 14679-14686,
15048-15055, 15417-15424, 15786-15793, 13203-13210, 13572-13579,
16155-16162, 16524-16531, 16893-16900, 17262-17269, 17631-17638,
18000-18007, 18369-18376, 18738-18745, 19107-19114, 19476-19483,
21363-21384, 21389-21410 or a fragment or variant of any of these
sequences.
18. Artificial RNA according to claim 15 to 17, wherein the at
least one coding sequence comprises a codon modified coding
sequence comprising a nucleic acid sequence being identical or at
least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:
70-71, 75-77, 485-486, 490-492, 899-900, 904-906, 1268-1269,
1273-1275, 1637-1638, 1642-1644, 2006-2007, 2011-2013, 2375-2376,
2380-2382, 2744-2745, 2749-2751, 3113-3114, 3118-3120, 3482-3483,
3487-3489, 3852, 4221, 4590, 4959, 5328, 5697, 6066, 6435, 6804,
7173, 7542, 7911, 3856-3858, 4225-4227, 4594-4596, 4963-4965,
5332-5334, 5701-5703, 6070-6072, 6439-6441, 6808-6810, 7177-7179,
7546-7548, 7915-7917, 11728, 11732-11734, 12097, 12101-12103,
12465, 12466, 12470-12472, 12834, 12835, 12839-12841, 13941, 13942,
13946-13948, 14310, 14311, 14315-14317, 14679, 14680, 14684-14686,
15048, 15049, 15053-15055, 15417, 15418, 15422-15424, 15786, 15787,
15791-15793, 13203, 13204, 13208-13210, 13572, 13573, 13577-13579,
16155, 16156, 16160-16162, 16524, 16525, 16529-16531, 16893, 16894,
16898-16900, 17262, 17263, 17267-17269, 17631, 17632, 17636-17638,
18000, 18001, 18005-18007, 18369, 18370, 18374-18376, 18738, 18739,
18743-18745, 19107, 19108, 19112-19114, 19476, 19477, 19481-19483,
21363-21384, 21389-21410 or a fragment or variant of any of these
sequences.
19. Artificial RNA according to any one of the preceding claims,
wherein the RNA is an mRNA, a self-replicating RNA, a circular RNA,
or a replicon RNA.
20. Artificial RNA according to claim 19, wherein the RNA is an
mRNA.
21. Artificial RNA according to any one of the preceding claims,
wherein the RNA comprises a 5'-cap structure, preferably m7G, cap0,
cap1, cap2, a modified cap0 or a modified cap1 structure.
22. Artificial RNA according to any one of the preceding claims,
wherein the RNA comprises at least one poly(A) sequence, preferably
comprising 30 to 150 adenosine nucleotides, more preferably
comprising 100 adenosine nucleotides and/or at least one poly(C)
sequence, preferably comprising 10 to 40 cytosine nucleotides.
23. Artificial RNA according to any one of the preceding claims,
wherein the RNA comprises at least one histone stem-loop, wherein
the histone stem-loop preferably comprises a nucleic acid sequence
according to SEQ ID NO: 39 or 40 or a fragment or variant
thereof.
24. Artificial RNA according to any one of the preceding claims
comprising the following elements, preferably in 5'- to
3'-direction: a) 5'-cap structure, preferably as specified herein;
b) optionally, 5'-UTR as specified herein, preferably at least one
selected from SEQ ID NOs: 1-22; c) a ribosome binding site,
preferably as specified herein d) at least one coding sequence as
specified herein, preferably as specified in Table 3 and Table 4;
d) 3'-UTR as specified herein, preferably at least one selected
from SEQ ID NOs: 23-38; e) optionally, a poly(A) sequence,
preferably as specified herein; f) optionally, a poly(C) sequence,
preferably as specified herein; g) optionally, a histone stem-loop,
preferably as specified herein; h) optionally, a 3'-terminal
sequence element as specified herein, preferably according to
according to SEQ ID NOs: 44-63, or 21322-21328;and wherein
optionally at least one or more than one, preferably wherein all
uracil nucleotides are replaced by pseudouridine (.psi.)
nucleotides or N1-methylpseudouridine (m1.psi.) nucleotides.
25. Artificial RNA according claims 1 to 24 comprising the
following elements, preferably in 5'- to 3-direction: a) 5'-cap
structure, preferably as defined in claim 21, most preferably a Cap
structure; b) 5'-UTR and/or 3'-UTR according to a-1, a-4, c-1, e-4,
g-2, i-2, or i-3; c) at least one coding sequence as defined by any
one of claims 13 to 18 or encoding a protein as defined by any one
of claims 7 to 12 or; d) optionally, a poly(A) sequence, preferably
as defined by claim 22; e) optionally, poly(C) sequence, preferably
as defined by claim 22; f) optionally, histone stem-loop,
preferably as defined by any one of claim 23; g) optionally, a
3'-terminal sequence according to SEQ ID NOs: 44-63, 21322-21328
and wherein optionally at least one or more than one, preferably
wherein all uracil nucleotides are replaced by pseudouridine
(.psi.) nucleotides or N1-methylpseudouridine (m1.psi.)
nucleotides.
26. Artificial RNA according claims 1 to 25 comprising the
following elements in 5'- to 3'-direction: a) 5'-cap structure,
preferably as defined in claim 21, most preferably a Cap1
structure; b) 5'-UTR and/or 3'-UTR according to a-1, a-4, c-1, e-4,
g-2, i-2, or i-3, preferably a-1; c) at least one coding sequence
as defined by any one of claims 13 to 18 or encoding a protein as
defined by any one of claims 7 to 12, wherein said coding sequence
is located between said 5'-UTR and said 3'-UTR, preferably
downstream of said 5'-UTR and upstream of said 3'-UTR; d) a histone
stem-loop, preferably as defined by any one of claim 23 e) a
poly(A) sequence, preferably as defined by claim 22 f) a
3'-terminal sequence according to SEQ ID NOs: 21322-21328. wherein
optionally at least one or more than one, preferably wherein all
uracil nucleotides are replaced by pseudouridine (.psi.)
nucleotides or N1-methylpseudouridine (m1.psi.) nucleotides.
27. Artificial RNA any one of the preceding claims, wherein the
artificial RNA comprises or consists of an RNA sequence which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373,
2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587,
4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801,
6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094,
12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677,
14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570,
13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629,
17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474,
19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 or
a fragment or variant of any of these sequences.
28. Artificial RNA according to claim 27, wherein said artificial
RNA comprises or consists of an RNA sequence which is identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 78-482,
11735-12094, 21415-21417, 21561-21563, 21489, 21490, 21635, 21636
or a fragment or variant of any of these sequences; said artificial
RNA comprises or consists of an RNA sequence which is identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 493-897,
12104-12463, 21418-21420, 21564-21566, 21491, 21492, 21637, 21638
or a fragment or variant of any of these sequences; said artificial
RNA comprises or consists of an RNA sequence which is identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs:
907-1266, 12473-12832, 21421-21423, 21567-21569, 21493-21495,
21639-21641 or a fragment or variant of any of these sequences;
said artificial RNA comprises or consists of an RNA sequence which
is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 1276-1635, 8278, 12842-13201, 21424-21426, 21570-21572,
21496-21498, 21642-21644 or a fragment or variant of any of these
sequences; said artificial RNA comprises or consists of an RNA
sequence which is identical or at least 70%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1645-2004, 13949-14308, 21433-21435,
21579-21581, 21505-21507, 21651-21653 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 2014-2373, 14318-14677, 21436-21438,
21582-21584, 21508-21510, 21654-21656 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 2383-2742, 14687-15046, 21439-21441,
21585-21587, 21511-21513, 21657-21659 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 2752-3111, 15056-15415, 21442-21444,
21588-21590, 21514-21516, 21660-21662 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 3121-3480, 15425-15784, 21445-21447,
21591-21593, 21517-21519, 21663-21665 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 3490-3849, 15794-16153, 1448-21450,
21594-21596, 21520-21522, 21666-21668 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 3859-4218, 13211-13570, 21427-21429,
21573-21575, 21499-21501, 21645-21647 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 4228-4587, 13580-13939, 21430-21432,
21576-21578, 21502-21504, 21648-21650 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 4597-4956, 16163-16522, 21451-21453,
21597-21599, 21523-21525, 21669-21671 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 4966-5325, 16532-16891, 21454-21456,
21600-21602, 21526-21528, 21672-21674 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 5335-5694, 16901-17260, 21457-21459,
21603-21605, 21529-21531, 21675-21677 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 5704-6063, 17270-17629, 21460-21462,
21606-21608, 21532-21534, 21678-21680 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 6073-6432, 17639-17998, 21463-21465,
21609-21611, 21535-21537, 21681-21683 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 6442-6801, 18008-18367, 21466-21468,
21612-21614, 21538-21540, 21684-21686 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 6811-7170, 18377-18736, 21469-21471,
21615-21617, 21541-21543, 21687-21689 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 7180-7539, 18746-19105, 21472-21474,
21618-21620, 21544-21546, 21690-21692 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 7549-7908, 19115-19474, 21475-21477,
21621-21623, 21547-21549, 21693-21695 or a fragment or variant of
any of these sequences; said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 7918-8277, 19484-19843, 21478-21480,
21624-21626, 21550-21552, 21696-21698 or a fragment or variant of
any of these sequences;
29. A composition comprising at least one artificial RNA as defined
in any one of claims 1 to 28, wherein the composition optionally
comprises at least one pharmaceutically acceptable carrier.
30. A composition according to claim 29 comprising at least one
further artificial RNA comprising at least one coding sequence
encoding at least one antigenic peptide or protein derived from RSV
selected from matrix protein M, nucleoprotein N, M2-1 protein,
and/or phosphoprotein P or combinations thereof, preferably
selected from M2-1.
31. A composition according to claim 29 or 30, comprising two
further artificial RNA species each comprising at least one coding
sequence encoding at least one antigenic peptide or protein derived
from RSV selected from matrix protein M, nucleoprotein N, M2-1, and
phosphoprotein P.
32. A composition according to claim 29 or 30, comprising three
further artificial RNA species each comprising at least one coding
sequence encoding at least one antigenic peptide or protein derived
from RSV selected from matrix protein M, nucleoprotein N, M2-1, and
phosphoprotein P.
33. A composition according to claims 30 to 32, wherein the coding
sequence of the further artificial RNA encodes at least one of the
amino acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to any one of SEQ ID NOs: 9684, 10053-10133, 10134,
10503-10636, 10637, 11006-11182, 11183, 11552-11725, 19844, 20213,
20582, 20951 or a fragment or variant of any of these
sequences.
34. A composition according to claim 30 to 33, wherein the coding
sequence is operably linked to a 3'-UTR and a 5'-UTR selected from
a-1, a-2, a-3, a-4, a-5, b-1, b-2, b-3, b-4, b-5, c-1, c-2, c-3,
c-4, c-5, d-1, d-2, d-3, d-4, d-5, e-1, e-2, e-3, e-4, e-5, e-6,
f-1, f-2, f-3, f-4, f-5, g-1, g-2, g-3, g-4, g-5, h-1, h-2, h-3,
h-4, h-5, i-1, i-2, or i-3.
35. A composition according to claim 30 to 34, wherein the coding
sequence comprises at least one of the nucleic acid sequences being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs:
9685-9692, 10135-10142, 10638-10645, 11184-11191, 19845-19852,
20214-20221, 20583-20590, 20952-20959, 21385-21388, 21411-21414 or
a fragment or a fragment or variant of any of these sequences.
36. A composition according to claim 30 to 35, wherein the further
artificial RNA comprises or consists of an RNA sequence which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from SEQ ID NOs: 9693-10052, 10143-10502,
10646-11005, 11192-11551, 19853-20212, 20222-20581, 20591-20950,
20960-21319, 21481-21634, 21553-21706 or a fragment or variant of
any of these sequences.
37. A composition according to claim 29 to 36 comprising an
artificial RNA which is identical or at least 70%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from SEQ ID NOs:
78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373,
2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587,
4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801,
6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094,
12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677,
14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570,
13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629,
17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474,
19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 and
a further artificial RNA which is identical or at least 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 9693-10052, 19853-20212, 21481,
21482, 21627, 21628, 21553, 21554, 21699, 21700; or an artificial
RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to a nucleic acid sequence selected from SEQ ID NOs: 78-482,
493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742,
2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956,
4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,
7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,
12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,
15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,
16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,
18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,
21415-21480, 21561-21626, 21489-21552, 21635-21698 and a further
artificial RNA which is identical or at least 70%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 10143-10502, 20222-20581, 21483, 21484,
21629, 21630, 21555, 21556, 21701, 21702; or an artificial RNA
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from SEQ ID NOs: 78-482, 493-897,
907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111,
3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325,
5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539,
7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832,
12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415,
15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522,
16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367,
18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480,
21561-21626, 21489-21552, 21635-21698 and a further artificial RNA
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 10646-11005, 20591-20950, 21485, 21486, 21631, 21632, 21557,
21558, 21703, 21704; or an artificial RNA which is identical or at
least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence
selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,
1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849,
3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063,
6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277,
8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201,
13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784,
15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891,
16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736,
18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626,
21489-21552, 21635-21698 and a further artificial RNA which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
11192-11551, 20960-21319; 21487, 21488, 21633, 21634, 21559, 21560,
21705, 21706 or an artificial RNA which is identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected
from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004,
2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218,
4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432,
6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278,
11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308,
14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153,
13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260,
17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105,
19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552,
21635-21698 and a further artificial RNA which is identical or at
least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence
selected from SEQ ID NOs: 9693-10052, 19853-20212, 21481, 21482,
21627, 21628, 21553, 21554, 21699, 21700 and a further artificial
RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to a nucleic acid sequence selected from SEQ ID NOs: 10646-11005,
20591-20950, 21485, 21486, 21631, 21632, 21557, 21558, 21703,
21704; or an artificial RNA which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004,
2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218,
4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432,
6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278,
11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308,
14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153,
13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260,
17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105,
19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552,
21635-21698 and a further artificial RNA which is identical or at
least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence
selected from SEQ ID NOs: 9693-10052, 19853-20212, 21481, 21482,
21627, 21628, 21553, 21554, 21699, 21700 and a further artificial
RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to a nucleic acid sequence selected from SEQ ID NOs: 10646-11005,
20591-20950, 21485, 21486, 21631, 21632, 21557, 21558, 21703, 21704
and a further artificial RNA which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
SEQ ID NOs: 10143-10502, 20222-20581, 21483, 21484, 21629, 21630,
21555, 21556, 21701, 21702.
38. Composition according to claim 29 to 37, wherein the at least
one artificial RNA and, optionally, the further artificial RNA is
complexed or associated with or at least partially complexed or
partially associated with one or more cationic or polycationic
compound, preferably cationic or polycationic polymer, cationic or
polycationic polysaccharide, cationic or polycationic lipid,
cationic or polycationic protein, cationic or polycationic peptide,
or any combinations thereof.
39. Composition according to claim 38, wherein the artificial RNA
and, optionally, the further artificial RNA is complexed or
associated with one or more lipids, thereby forming liposomes,
lipid nanoparticles, lipoplexes, and/or nanoliposomes.
40. Composition according to claim 39, wherein the artificial RNA
and, optionally, the further artificial RNA is complexed with one
or more lipids thereby forming lipid nanoparticles (LNP).
41. Composition according to claim 40, wherein the LNP comprises a
cationic lipid with the formula Ill: ##STR00056## or a
pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer
thereof, wherein: L.sup.1 or L.sup.2 is each independently
--O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O)--,
--S--S--, --C(.dbd.O)S--, --SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)NR.sup.a--,
--OC(.dbd.O)NR.sup.a-- or --NR.sup.aC(.dbd.O)O--, preferably
L.sup.1 or L.sup.2 is --O(C.dbd.O)-- or --(C.dbd.O).theta.-;
G.sup.1 and G.sup.2 are each independently unsubstituted
C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; G.sup.3
is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24 alkenylene,
C.sub.3-C.sub.8 cycloalkylene, or C.sub.3-C.sub.8 cycloalkenylene;
R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.1 and R.sup.2 are
each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24
alkenyl; R.sup.3 is H, OR.sup.5, CN, --C(.dbd.O)OR.sup.4,
--OC(.dbd.O)R.sup.4 or --NR.sup.5C(.dbd.O)R.sup.4; R.sup.4 is
C.sub.1-C.sub.12 alkyl; R.sup.5 is H or C.sub.1-C.sub.12 alkyl; and
x is 0, 1 or 2;
42. Composition according to claim 41, wherein the cationic lipid
is a compound of formula I, and wherein: L.sup.1 and L.sup.2 are
each independently --O(C.dbd.O)-- or (C.dbd.O)--O--; G.sup.3 is
C.sub.1-C.sub.24 alkylene or C.sub.1-C.sub.24 alkenylene; and
R.sup.3 is H or OR.sup.5.
43. Composition according to any one of claims 41 to 42, wherein
the cationic lipid is a compound of formula III, and wherein:
L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)-- or
(C.dbd.O)--O--; and R.sup.1 and R.sup.2 each independently have one
of the following structures: ##STR00057##
44. Composition according to any one of claims 41 to 43, wherein
the cationic lipid is a compound of formula III, and wherein
R.sup.3 is OH.
45. Composition according to any one of claims 41 to 44, wherein
the cationic lipid is selected from structures III-1 to III-36:
TABLE-US-00026 No. Structure III-1 ##STR00058## III-2 ##STR00059##
III-3 ##STR00060## III-4 ##STR00061## III-5 ##STR00062## III-6
##STR00063## III-7 ##STR00064## III-8 ##STR00065## III-9
##STR00066## III-10 ##STR00067## III-11 ##STR00068## III-12
##STR00069## III-13 ##STR00070## III-14 ##STR00071## III-15
##STR00072## III-16 ##STR00073## III-17 ##STR00074## III-18
##STR00075## III-19 ##STR00076## III-20 ##STR00077## III-21
##STR00078## III-22 ##STR00079## III-23 ##STR00080## III-24
##STR00081## III-25 ##STR00082## III-26 ##STR00083## III-27
##STR00084## III-28 ##STR00085## III-29 ##STR00086## III-30
##STR00087## III-31 ##STR00088## III-32 ##STR00089## III-33
##STR00090## III-34 ##STR00091## III-35 ##STR00092## III-36
##STR00093##
46. Composition according to any one of claims 41 to 45, wherein
the cationic lipid is ##STR00094##
47. Composition according to any one of claims 40 to 46, wherein
the LNP comprises a PEG lipid with the formula (IV): ##STR00095##
wherein R.sup.8 and R.sup.9 are each independently a straight or
branched, saturated or unsaturated alkyl chain containing from 10
to 30 carbon atoms, wherein the alkyl chain is optionally
interrupted by one or more ester bonds; and w has a mean value
ranging from 30 to 60.
48. Composition according to claim 47, wherein in the PEG lipid
R.sup.8 and R.sup.9 are saturated alkyl chains.
49. Composition according to claim 47 or 48, wherein the PEG lipid
is ##STR00096## wherein n has a mean value ranging from 30 to 60,
preferably wherein n has a mean value of about 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, most preferably wherein n has a mean value of
49.
50. Composition according to any one of claims 40 to 49, wherein
the LNP comprises one or more neutral lipids and/or a steroid or
steroid analogues.
51. Composition according to claim 50, wherein the neutral lipid is
selected from the group comprising distearoylphosphatidylcholine
(DSPC), dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol
(DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
52. Composition according to claim 50 or 51, wherein the neutral
lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and
wherein the molar ratio of the cationic lipid to DSPC is optionally
in the range from about 2:1 to 8:1.
53. Composition according to claim 50, wherein the steroid is
cholesterol, and wherein the molar ratio of the cationic lipid to
cholesterol is optionally in the range from about 2:1 to 1:1
54. Composition according to any one of claims 40 to 53, wherein
the LNP essentially consists of (i) at least one cationic lipid,
preferably as defined in any one of claims 41 to 46; (ii) a neutral
lipid, preferably as defined in any one of claims 50 to 52; (iii) a
steroid or steroid analogue, preferably as defined in claim 53; and
(iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, preferably as defined
in any one of claims 47 to 49, wherein (i) to (iv) are in a molar
ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55%
sterol, and 0.5-15% PEG-lipid.
55. Polypeptide encoded by the artificial RNA according to any one
of claims 1 to 28, preferably having an amino acid sequence which
is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID
NOs: 1267, 2005, 2743, 3481, 4219, 4957, 5695, 6433, 7171, 7909,
12833, 14309, 15047, 15785, 13571, 16523, 17261, 17999, 18737,
19475 or a variant of any of these polypeptides.
56. A composition comprising the polypeptide as defined in claim
55, wherein the composition optionally comprises at least one
pharmaceutically acceptable carrier or at least one artificial RNA
as defined in any one of claims 1 to 28, or a composition as
defined in any one of claims 29 to 54.
57. A vaccine comprising the artificial RNA as defined in any one
of claims 1 to 28, the composition as defined in any one of claims
29 to 54, the polypeptide as defined in claim 55, or the
composition as defined in claim 56.
58. Vaccine according to claim 49, wherein the artificial RNA as
defined in any one of claims 1 to 28, the composition as defined in
any one of claims 29 to 54, the polypeptide as defined in claim 55,
or the composition as defined in claim 56 elicits an adaptive
immune response.
59. Vaccine according to claim 57 or 58, wherein the vaccine
further comprises a pharmaceutically acceptable carrier and
optionally at least one adjuvant.
60. A Kit or kit of parts comprising the artificial RNA as defined
in any one of claims 1 to 28, the composition as defined in any one
of claims 29 to 54, the polypeptide as defined in claim 55, the
composition as defined in claim 56, and/or the vaccine as defined
in any one of claims 57 to 59, optionally comprising a liquid
vehicle for solubilising, and optionally technical instructions
providing information on administration and dosage of the
components.
61. Kit or kit of parts according to claim 60 comprising at least
the following components a) at least one artificial RNA as defined
in any one of claims 1 to 28 encoding at least one antigenic
peptide or protein derived from a RSV fusion (F) protein, wherein
said artificial RNA is preferably complexed with one or more lipids
thereby forming lipid nanoparticles (LNP); and b) at least one, two
or three further artificial RNA species each encoding an antigenic
peptide or protein derived from RSV selected from M, N, M2-1, or P,
wherein each of said further artificial RNA species are preferably
complexed with one or more lipids thereby forming lipid
nanoparticles (LNP), wherein components a) and b) are provided as
separate entities or as a single entity.
62. Kit or kit of parts according to claim 60 or 61 further
comprising Ringer lactate solution.
63. Artificial RNA as defined in any one of claims 1 to 28, the
composition as defined in any one of claims 29 to 54, the
polypeptide as defined in claim 55, the composition as defined in
claim 56, the vaccine as defined in any one of claims 57 to 59, or
the kit or kit of parts as defined in claim 60 to 62 for use as a
medicament.
64. Artificial RNA as defined in any one of claims 1 to 28, the
composition as defined in any one of claims 29 to 54, the
polypeptide as defined in claim 55, the composition as defined in
claim 56, the vaccine as defined in any one of claims 57 to 59, or
the kit or kit of parts as defined in claim 60 to 62 for use in the
treatment or prophylaxis of an infection with a virus, preferably
with RSV, or a disorder related to such an infection.
65. A method of treating or preventing a disorder, wherein the
method comprises applying or administering to a subject in need
thereof the artificial RNA as defined in any one of claims 1 to 28,
the composition as defined in any one of claims 29 to 54, the
polypeptide as defined in claim 55, the composition as defined in
claim 56, the vaccine as defined in any one of claims 57 to 59, or
the kit or kit of parts as defined in claim 60 to 62
66. Method according to claim 65, wherein the disorder is an
infection with a RSV, or a disorder related to such an
infection.
67. Method according to claim 65 or 66, wherein the subject in need
is a mammalian subject.
68. Method according to claim 67, wherein the mammalian subject is
a human subject, preferably a newborn, a pregnant woman, a
breast-feeding woman, an elderly, and/or an immunocompromised human
subject
Description
INTRODUCTION
[0001] The present invention is directed to artificial RNA suitable
for use in the treatment or prophylaxis of an infection with
Respiratory syncytial virus (RSV) or of a disorder related to such
an infection. In particular, the artificial RNA of the invention
comprises at least one heterologous untranslated region (UTR),
preferably a 3'-UTR and/or a 5'-UTR, and a coding region encoding
at least one antigenic peptide or protein derived from RSV, in
particular at least one antigenic peptide or protein derived from
RSV fusion (F) protein. The artificial RNA is preferably
characterized by increased expression efficacies of coding regions
operably linked to said UTR elements. The present invention is also
directed to compositions and vaccines comprising said artificial
RNA in association with a polymeric carrier, a polycationic protein
or peptide, or a lipid nanoparticle (LNP). Further, the invention
concerns a kit, particularly a kit of parts comprising the
artificial RNA or composition or vaccine. The invention is further
directed to a method of treating or preventing a disorder or a
disease, and first and second medical uses of the artificial RNA,
composition, or vaccine.
[0002] Respiratory syncytial virus (RSV) is an enveloped
non-segmented negative-strand RNA virus in the family
Paramyxoviridae, genus Pneumovirus. It is the most common cause of
bronchiolitis and pneumonia among children in their first year of
life. RSV also causes repeated infections including severe lower
respiratory tract disease, which may occur at any age, especially
among the elderly or those with compromised cardiac, pulmonary, or
immune systems. Currently, passive immunization is used to prevent
severe illness caused by RSV infection, especially in infants with
prematurity, bronchopulmonary dysplasia, or congenital heart
disease.
[0003] Recommended treatment of RSV bronchiolitis primarily
consists of respiratory support and hydration. No specific
anti-viral therapy is recommended. The neutralizing monoclonal
antibody Palivizumab is used for prophylaxis of infants at highest
risk for severe infection but is too expensive and impractical for
universal use. Currently, there is no licensed/approved RSV
vaccine, and developing a safe and effective RSV vaccine is a
global public health priority.
[0004] In a vaccine trial in the 1960s, infants and young children
were immunized with a formalin-inactivated whole virion RSV
preparation (FIRSV) or an equivalent paramyxovirus preparation
(FIPIV). Five percent of the subjects who were immunized with F-PIV
and then naturally infected by RSV during the next RSV season were
hospitalized; 80% of those who were immunized with FI-RSV and then
infected by RSV were hospitalized, and two children died. This
enhancement of an RSV infection due to vaccination is a specific
problem for the development of vaccines against RSV infections.
[0005] Therefore, Respiratory syncytial virus (RSV) infections are
the greatest remaining unmet infant vaccine need in developed
countries and an important unmet infant vaccine need worldwide.
More than 40 years of effort have not yet resulted in a licensed
RSV vaccine for humans.
[0006] Despite the above mentioned humanized monoclonal antibody
Palivizumab, live-attenuated vaccine viruses were developed which
elicit a strong immune response, but which are not recommended for
use in the specific target groups (infants, children, the elderly
and immunocompromised patients). Also, DNA vectors expressing RSV F
protein which bears B-cell epitopes were used to induce the
production of neutralizing antibodies. In this context,
WO2008/077527 and WO96/040945 disclose vectors comprising DNA
sequences encoding RSV F protein for the use as vaccines. However,
the use of DNA as a vaccine may be dangerous due to unwanted
insertion into the genome, possibly leading to interruption of
functional genes and cancer or the formation of anti-DNA
antibodies.
[0007] WO2015/024668 discloses RNA sequences encoding RSV antigenic
peptides and proteins selected from fusion protein F, the
glycoprotein G, the short hydrophobic protein SH, the matrix
protein M, the nucleoprotein N, the large polymerase L, the M2-1
protein, the M2-2 protein, the phosphoprotein P, the non-structural
protein NS1 or the non-structural protein NS2, and an antigenic
composition comprising protamine-complexed RNA suitable for
intradermal administration.
[0008] WO2017/070622 discloses a vaccine comprising RNA encoding
RSV antigenic peptides and proteins selected from glycoprotein F
and glycoprotein G, wherein the RNA is formulated in lipid
nanoparticles.
[0009] Apart from some approaches cited above, there remains an
unmet medical need for an efficient vaccine for prophylaxis or
treatment of RSV infections.
[0010] Accordingly, it is the object of the underlying invention to
provide novel artificial RNA coding for antigenic peptides or
proteins of RSV and compositions/vaccines comprising said RNA for
the use as vaccine for prophylaxis or treatment of RSV infections,
particularly in infants, newborns, pregnant women, elderly, and
immunocompromised patients.
[0011] Further it would be desirable that an RNA-based composition
or vaccine has some of the following advantageous features: [0012]
Improved translation of RNA constructs at the site of injection
(e.g. muscle) [0013] Very efficient induction of RSV
antigen-specific immune responses against the encoded antigenic
peptide or protein at a very low dosages and dosing regimen. [0014]
Suitability for maternal immunization [0015] Suitability for
vaccination of infants and/or newborns [0016] Suitability for
intramuscular administration [0017] Induction of an RSV-specific
functional humoral immune response [0018] Induction of RSV-specific
B-cell memory [0019] Faster onset of immune protection against RSV
[0020] Longevity of the induced immune responses against RSV [0021]
Induction of broad cellular T-cell responses against RSV [0022]
Induction of a (local and transient) pro-inflammatory environment
[0023] No induction of systemic cytokine or chemokine response
after application of the vaccine [0024] Well tolerability, no
side-effects, non toxic, [0025] No enhancement of an RSV infection
due to vaccination [0026] Advantageous stability characteristics of
the vaccine [0027] Speed, adaptability, simplicity and scalability
of RSV vaccine production
[0028] The objects outlined above are solved by the claimed subject
matter.
Definitions
[0029] For the sake of clarity and readability the following
definitions are provided. Any technical feature mentioned for these
definitions may be read on each and every embodiment of the
invention. Additional definitions and explanations may be
specifically provided in the context of these embodiments.
[0030] Percentages in the context of numbers should be understood
as relative to the total number of the respective items. In other
cases, and unless the context dictates otherwise, percentages
should be understood as percentages by weight (wt.-%).
[0031] Adaptive immune response: The term "adaptive immune
response" as used herein will be recognized and understood by the
person of ordinary skill in the art, and is for example intended to
refer to an antigen-specific response of the immune system (the
adaptive immune system). Antigen specificity allows for the
generation of responses that are tailored to specific pathogens or
pathogen-infected cells. The ability to mount these tailored
responses is usually maintained in the body by "memory cells"
(B-cells). In the context of the invention, the antigen is provided
by the artificial RNA coding sequence encoding at least one
antigenic peptide or protein.
[0032] Antigen: The term "antigen" as used herein will be
recognized and understood by the person of ordinary skill in the
art, and is for example intended to refer to a substance which may
be recognized by the immune system, preferably by the adaptive
immune system, and is capable of triggering an antigen-specific
immune response, e.g. by formation of antibodies and/or
antigen-specific T cells as part of an adaptive immune response.
Typically, an antigen may be or may comprise a peptide or protein
which may be presented by the MHC to T-cells. Also fragments,
variants and derivatives of peptides or proteins derived from e.g.
RSV F protein comprising at least one epitope are understood as
antigens in the context of the invention. In the context of the
present invention, an antigen may be the product of translation of
a provided artificial RNA as specified herein.
[0033] Antigenic peptide or protein: The term "antigenic peptide or
protein" will be recognized and understood by the person of
ordinary skill in the art, and is for example intended to refer to
a peptide, protein (or polyprotein) derived from a (antigenic)
protein/polyprotein which may stimulate the body's adaptive immune
system to provide an adaptive immune response. Therefore an
"antigenic peptide or protein" comprises at least one epitope (as
defined herein) or antigen (as defined herein) of the protein it is
derived from (e.g., in the context of the invention, RSV peptide or
protein, preferably RSV F protein or variants thereof).
[0034] Artificial nucleic acid: The terms "artificial nucleic acid"
as used herein will be recognized and understood by the person of
ordinary skill in the art, and are for example intended to refer to
an artificial nucleic acid that does not occur naturally. An
artificial nucleic acid may be a DNA molecule, an RNA molecule or a
hybrid-molecule comprising DNA and RNA portions. Typically,
artificial nucleic acids may be designed and/or generated by
genetic engineering methods to correspond to a desired artificial
sequence of nucleotides (heterologous sequence). In this context an
artificial sequence is usually a sequence that may not occur
naturally, i.e. it differs from the wild type sequence by at least
one nucleotide. The term "wild type" as used herein will be
recognized and understood by the person of ordinary skill in the
art, and is for example intended to refer to a sequence occurring
in nature. Further, the term "artificial nucleic acid" is not
restricted to mean "one single molecule" but is, typically,
understood to comprise an ensemble of essentially identical
molecules.
[0035] Artificial RNA: The term "artificial RNA" as used herein is
intended to refer to an RNA that does not occur naturally. In other
words, an artificial RNA may be understood as a non-natural nucleic
acid molecule. Such RNA molecules may be non-natural due to its
individual sequence (which does not occur naturally, e.g. G/C
content modified coding sequence, UTRs) and/or due to other
modifications, e.g. structural modifications of nucleotides which
do not occur naturally. Typically, artificial RNA may be designed
and/or generated by genetic engineering methods to correspond to a
desired artificial sequence of nucleotides (heterologous sequence).
In this context an artificial RNA sequence is usually a sequence
that may not occur naturally, i.e. it differs from the wild type
sequence by at least one nucleotide. The term "artificial RNA" is
not restricted to mean "one single molecule" but is, typically,
understood to comprise an ensemble of essentially identical
molecules. Accordingly, it may relate to a plurality of essentially
identical RNA molecules contained in an aliquot or a sample. In the
context of the invention, the RNA of the invention is an artificial
RNA as defined herein.
[0036] Cationic: Unless a different meaning is clear from the
specific context, the term "cationic" means that the respective
structure bears a positive charge, either permanently or not
permanently but in response to certain conditions such as pH. Thus,
the term "cationic" covers both "permanently cationic" and
"cationisable".
[0037] Cationisable: The term "cationisable" as used herein means
that a compound, or group or atom, is positively charged at a lower
pH and uncharged at a higher pH of its environment. Also in
non-aqueous environments where no pH value can be determined, a
cationisable compound, group or atom is positively charged at a
high hydrogen ion concentration and uncharged at a low
concentration or activity of hydrogen ions. It depends on the
individual properties of the cationisable or polycationisable
compound, in particular the pKa of the respective cationisable
group or atom, at which pH or hydrogen ion concentration it is
charged or uncharged. In diluted aqueous environments, the fraction
of cationisable compounds, groups or atoms bearing a positive
charge may be estimated using the so-called Henderson-Hasselbalch
equation which is well-known to a person skilled in the art. For
example, in some embodiments, if a compound or moiety is
cationisable, it is preferred that it is positively charged at a pH
value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8,
more preferably of a pH value of or below 9, of or below 8, of or
below 7, most preferably at physiological pH values, e.g. about 7.3
to 7.4, i.e. under physiological conditions, particularly under
physiological salt conditions of the cell in vivo. In other
embodiments, it is preferred that the cationisable compound or
moiety is predominantly neutral at physiological pH values, e.g.
about 7.0-7.4, but becomes positively charged at lower pH values.
In some embodiments, the preferred range of pKa for the
cationisable compound or moiety is about 5 to about 7.
[0038] Coding sequence/coding region: The terms "coding sequence"
or "coding region" and the corresponding abbreviation "cds" as used
herein will be recognized and understood by the person of ordinary
skill in the art, and are for example intended to refer to a
sequence of several nucleotide triplets, which may be translated
into a peptide or protein. A coding sequence in the context of the
present invention is preferably an RNA sequence, consisting of a
number of nucleotides that may be divided by three, which starts
with a start codon and which preferably terminates with a stop
codon.
[0039] Composition: In the context of the invention, a
"composition" refers to any type of composition in which the
specified ingredients (e.g. artificial RNA of the invention in
association with LNP), may be incorporated, optionally along with
any further constituents, usually with at least one
pharmaceutically acceptable carrier or excipient. Thus, the
composition may be a dry composition such as a powder or granules,
or a solid unit such as a lyophilized form or a tablet.
Alternatively, the composition may be in liquid form, and each
constituent may be independently incorporated in dissolved or
dispersed (e.g. suspended or emulsified) form.
[0040] Compound: As used herein, a "compound" means a chemical
substance, which is a material consisting of molecules having
essentially the same chemical structure and properties. For a small
molecular compound, the molecules are typically identical with
respect to their atomic composition and structural configuration.
For a macromolecular or polymeric compound, the molecules of a
compound are highly similar but not all of them are necessarily
identical. For example, a segment of a polymer that is designated
to consist of 50 monomeric units may also contain individual
molecules with e.g. 48 or 53 monomeric units.
[0041] Derived from: The term "derived from" as used throughout the
present specification in the context of a nucleic acid, i.e. for a
nucleic acid "derived from" (another) nucleic acid, means that the
nucleic acid, which is derived from (another) nucleic acid, shares
at least 50%, preferably at least 55%, preferably at least 60%,
preferably at least 65%, preferably at least 70%, more preferably
at least 75%, more preferably at least 80%, 81%, 82%, 83%, 84%,
more preferably at least 85%, 86%, 87%, 88%, 89% even more
preferably at least 90%, 91%, 92%, 93%, 94%, even more preferably
at least 95%, 96%, 97%, and particularly preferably at least 98%,
99% sequence identity with the nucleic acid from which it is
derived. The skilled person is aware that sequence identity is
typically calculated for the same types of nucleic acids, i.e. for
DNA sequences or for RNA sequences. Thus, it is understood, if a
DNA is "derived from" an RNA or if an RNA is "derived from" a DNA,
in a first step the RNA sequence is converted into the
corresponding DNA sequence (in particular by replacing the uracils
(U) by thymidines (T) throughout the sequence) or, vice versa, the
DNA sequence is converted into the corresponding RNA sequence (in
particular by replacing the thymidines (T) by uracils (U)
throughout the sequence). Thereafter, the sequence identity of the
DNA sequences or the sequence identity of the RNA sequences is
determined.
[0042] Preferably, a nucleic acid "derived from" a nucleic acid
also refers to nucleic acid, which is modified in comparison to the
nucleic acid from which it is derived, e.g. in order to increase
RNA stability even further and/or to prolong and/or increase
protein production. It goes without saying that such modifications
are preferred, which do not impair RNA stability, e.g. in
comparison to the nucleic acid from which it is derived. In the
context of amino acid sequences (e.g. antigenic peptides or
proteins) the term "derived from" means that the amino acid
sequence, which is derived from (another) amino acid sequence (e.g.
RSV F protein), shares at least 50%, preferably at least 55%,
preferably at least 60%, preferably at least 65%, preferably at
least 70%, more preferably at least 75%, more preferably at least
80%, 81%, 82%, 83%, 84%, more preferably at least 85%, 86%, 87%,
88%, 89% even more preferably at least 90%, 91%, 92%, 93%, 94%,
even more preferably at least 95%, 96%, 97%, and particularly
preferably at least 98%, 99% sequence identity with the amino acid
sequence from which it is derived.
[0043] Thus, it is understood, if a antigenic peptides or protein
is "derived from" an RSV fusion (F) protein, the antigenic peptides
or protein that is "derived from" said RSV F protein may represent
a variant or fragment of the RSV F protein, e.g. F0 (full-length
precursor), F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2,
F_DSCav1_mut3, F-del_DSCav1, F-del_DSCav1_mut1, F-del_DSCav1_mut2,
F-del_DSCav1_mut3 (as specified herein). Moreover, the antigenic
peptides or protein that is "derived from" said RSV F proteins
(e.g., F0, F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2,
F_DSCav1_mut3, F-del_DSCav1, F-del_DSCav1_mut1, F-del_DSCav1_mut2,
F-del_DSCav1_mut3) may differ in the amino acid sequence, sharing a
certain percentage of identity as defined above. Suitable further
examples of RSV F proteins from which an antigenic peptides or
protein may be "derived from" are provided in Table 1.
[0044] Epitope: The term "epitope" (also called "antigen
determinant" in the art) as used herein will be recognized and
understood by the person of ordinary skill in the art, and is for
example intended to refer to T cell epitopes and B cell epitopes. T
cell epitopes or parts of the antigenic peptides or proteins may
comprise fragments preferably having a length of about 6 to about
20 or even more amino acids, e.g. fragments as processed and
presented by MHC class I molecules, preferably having a length of
about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or
12 amino acids), or fragments as processed and presented by MHC
class II molecules, preferably having a length of about 13 to about
20 or even more amino acids, wherein these fragments may be
selected from any part of the amino acid sequence. These fragments
are typically recognized by T cells in form of a complex consisting
of the peptide fragment and an MHC molecule, i.e. the fragments are
typically not recognized in their native form. B cell epitopes are
typically fragments located on the outer surface of (native)
protein or peptide antigens, preferably having 5 to 15 amino acids,
more preferably having 5 to 12 amino acids, even more preferably
having 6 to 9 amino acids, which may be recognized by antibodies,
i.e. in their native form. Such epitopes of proteins or peptides
may furthermore be selected from any of the herein mentioned
variants of such proteins or peptides. In this context antigenic
determinants can be conformational or discontinuous epitopes which
are composed of segments of the proteins or peptides as defined
herein that are discontinuous in the amino acid sequence of the
proteins or peptides as defined herein but are brought together in
the three-dimensional structure or continuous or linear epitopes
which are composed of a single polypeptide chain. In the context of
the present invention, an epitope may be the product of translation
of a provided artificial RNA as specified herein.
[0045] Fragment: The term "fragment" as used throughout the present
specification in the context of a nucleic acid sequence or an amino
acid sequence may typically be a shorter portion of a full-length
sequence of e.g. a nucleic acid sequence or an amino acid sequence.
Accordingly, a fragment, typically, consists of a sequence that is
identical to the corresponding stretch within the full-length
sequence. A preferred fragment of a sequence in the context of the
present invention, consists of a continuous stretch of entities,
such as nucleotides or amino acids corresponding to a continuous
stretch of entities in the molecule the fragment is derived from,
which represents at least 5%, 10%, 20%, preferably at least 30%,
more preferably at least 40%, more preferably at least 50%, even
more preferably at least 60%, even more preferably at least 70%,
and most preferably at least 80% of the total (i.e. full-length)
molecule from which the fragment is derived (e.g. RSV F protein).
The term "fragment" as used throughout the present specification in
the context of proteins or peptides may, typically, comprise a
sequence of a protein or peptide as defined herein, which is, with
regard to its amino acid sequence (or its encoded nucleic acid
molecule), N-terminally and/or C-terminally truncated compared to
the amino acid sequence of the original (native) protein (or its
encoded nucleic acid molecule). Such truncation may thus occur
either on the amino acid level or correspondingly on the nucleic
acid level. A sequence identity with respect to such a fragment as
defined herein may therefore preferably refer to the entire protein
or peptide as defined herein or to the entire (coding) nucleic acid
molecule of such a protein or peptide. In the context of antigens
such fragment may have a length of about 6 to about 20 or even more
amino acids, e.g. fragments as processed and presented by MHC class
I molecules, preferably having a length of about 8 to about 10
amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino
acids), or fragments as processed and presented by MHC class I
molecules, preferably having a length of about 13 or more amino
acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino
acids, wherein these fragments may be selected from any part of the
amino acid sequence. These fragments are typically recognized by
T-cells in form of a complex consisting of the peptide fragment and
an MHC molecule, i.e. the fragments are typically not recognized in
their native form. Fragments of proteins or peptides (e.g. in the
context of antigens) may comprise at least one epitope of those
proteins or peptides. Furthermore also domains of a protein, like
the extracellular domain, the intracellular domain or the
transmembrane domain and shortened or truncated versions of a
protein may be understood to comprise a fragment of a protein.
[0046] Heterologous: The terms "heterologous" or "heterologous
sequence" as used throughout the present specification in the
context of a nucleic acid sequence or an amino acid sequence refers
to a sequence (e.g. DNA, RNA, amino acid) will be recognized and
understood by the person of ordinary skill in the art, and is
intended to refer to a sequence that is derived from another gene,
from another allele, from another species. Two sequences are
typically understood to be "heterologous" if they are not derivable
from the same gene or in the same allele. I.e., although
heterologous sequences may be derivable from the same organism,
they naturally (in nature) do not occur in the same nucleic acid
molecule, such as e.g. in the same RNA, or the same protein.
[0047] Humoral immune response: The terms "humoral immunity" or
"humoral immune response" will be recognized and understood by the
person of ordinary skill in the art, and are for example intended
to refer to B-cell mediated antibody production and optionally to
accessory processes accompanying antibody production. A humoral
immune response may be typically characterized, e.g. by Th2
activation and cytokine production, germinal center formation and
isotype switching, affinity maturation and memory cell generation.
Humoral immunity also typically may refer to the effector functions
of antibodies, which include pathogen and toxin neutralization,
classical complement activation, and opsonin promotion of
phagocytosis and pathogen elimination.
[0048] Identity (of a sequence): The term "identity" as used
throughout the present specification in the context of a nucleic
acid sequence or an amino acid sequence will be recognized and
understood by the person of ordinary skill in the art, and is for
example intended to refer to the percentage to which two sequences
are identical. To determine the percentage to which two sequences
are identical, e.g. nucleic acid sequences or amino acid sequences
as defined herein, preferably the amino acid sequences encoded by
the artificial nucleic acid sequence as defined herein or the amino
acid sequences themselves, the sequences can be aligned in order to
be subsequently compared to one another. Therefore, e.g. a position
of a first sequence may be compared with the corresponding position
of the second sequence. If a position in the first sequence is
occupied by the same component (residue) as is the case at a
position in the second sequence, the two sequences are identical at
this position. If this is not the case, the sequences differ at
this position. If insertions occur in the second sequence in
comparison to the first sequence, gaps can be inserted into the
first sequence to allow a further alignment. If deletions occur in
the second sequence in comparison to the first sequence, gaps can
be inserted into the second sequence to allow a further alignment.
The percentage to which two sequences are identical is then a
function of the number of identical positions divided by the total
number of positions including those positions which are only
occupied in one sequence. The percentage to which two sequences are
identical can be determined using a mathematical algorithm. A
preferred, but not limiting, example of a mathematical algorithm
which can be used is the algorithm is integrated in the BLAST
program. Sequences which are identical to the sequences of the
present invention to a certain extent can be identified by this
program.
[0049] Immunogen, immunogenic: The terms "immunogen" or
"immunogenic" will be recognized and understood by the person of
ordinary skill in the art, and are for example intended to refer to
a compound that is able to stimulate/induce an immune response.
Preferably, an immunogen is a peptide, polypeptide, or protein. An
immunogen in the sense of the present invention is the product of
translation of a provided artificial nucleic acid, preferably RNA,
comprising at least one coding sequence encoding at least one
antigenic peptide, protein derived from RSV as defined herein.
Typically, an immunogen elicits an adaptive immune response.
[0050] Immune response: The term "immune response" will be
recognized and understood by the person of ordinary skill in the
art, and is for example intended to refer to a specific reaction of
the adaptive immune system to a particular antigen (so called
specific or adaptive immune response) or an unspecific reaction of
the innate immune system (so called unspecific or innate immune
response), or a combination thereof.
[0051] Immune system: The term "immune system" will be recognized
and understood by the person of ordinary skill in the art, and is
for example intended to refer to a system of the organism that may
protect the organisms from infection. If a pathogen succeeds in
passing a physical barrier of an organism and enters this organism,
the innate immune system provides an immediate, but non-specific
response. If pathogens evade this innate response, vertebrates
possess a second layer of protection, the adaptive immune system.
Here, the immune system adapts its response during an infection to
improve its recognition of the pathogen. This improved response is
then retained after the pathogen has been eliminated, in the form
of an immunological memory, and allows the adaptive immune system
to mount faster and stronger attacks each time this pathogen is
encountered. According to this, the immune system comprises the
innate and the adaptive immune system. Each of these two parts
typically contains so called humoral and cellular components.
[0052] Innate immune system: The term "innate immune system" (also
known as non-specific or unspecific immune system) will be
recognized and understood by the person of ordinary skill in the
art, and is for example intended to refer to a system typically
comprising the cells and mechanisms that defend the host from
infection by other organisms in a non-specific manner. This means
that the cells of the innate system may recognize and respond to
pathogens in a generic way, but unlike the adaptive immune system,
it does not confer long-lasting or protective immunity to the host.
The innate immune system may be, e.g. activated by ligands of
Toll-like receptors (TLRs) or other auxiliary substances such as
lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines,
monokines, lymphokines, interleukins or chemokines, IL-1 to IL-33,
IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta,
TNF-alpha, growth factors, and hGH, a ligand of human Toll-like
receptor (e.g., TLR1 to TLR10), a ligand of murine Toll-like
receptor, (e.g., TLR1 to TLR13), a ligand of a NOD-like receptor, a
ligand of a RIG-I like receptor, an immunostimulatory nucleic acid,
an immunostimulatory RNA (isRNA), a CpG-DNA, an antibacterial
agent, or an anti-viral agent.
[0053] Lipidoid compound: A lipidoid compound, also simply referred
to as lipidoid, is a lipid-like compound, i.e. an amphiphilic
compound with lipid-like physical properties. In the context of the
present invention the term lipid is considered to encompass
lipidoid compounds.
[0054] Monovalent vaccine, monovalent composition: The terms
"monovalent vaccine", "monovalent composition" "univalent vaccine"
or "univalent composition" will be recognized and understood by the
person of ordinary skill in the art, and are for example intended
to refer to a composition or a vaccine comprising only one antigen
from a virus. Accordingly, said vaccine or composition comprises
only one RNA species encoding a single antigen for a single
organism. The term "monovalent vaccine" includes the immunization
against a single valence. In the context of the invention, a
monovalent RSV vaccine or composition would comprise an artificial
RNA encoding one single antigenic peptide or protein derived from
one specific RSV (e.g. RSV F).
[0055] Nucleic acid: The terms "nucleic acid" or "nucleic acid
molecule" will be recognized and understood by the person of
ordinary skill in the art, and are for example intended to refer to
a molecule comprising, preferably consisting of nucleic acid
components. The term nucleic acid molecule preferably refers to DNA
or RNA molecules. It is preferably used synonymous with the term
polynucleotide. Preferably, a nucleic acid or a nucleic acid
molecule is a polymer comprising or consisting of nucleotide
monomers, which are covalently linked to each other by
phosphodiester-bonds of a sugar/phosphate-backbone. The term
"nucleic acid molecule" also encompasses modified nucleic acid
molecules, such as base-modified, sugar-modified or
backbone-modified DNA or RNA molecules as defined herein.
[0056] Nucleic acid sequence/RNA sequence/amino acid sequence: The
terms "nucleic acid sequence", "RNA sequence" or "amino acid
sequence" will be recognized and understood by the person of
ordinary skill in the art, and are for example intended to refer to
particular and individual order of the succession of its
nucleotides or amino acids respectively.
[0057] Permanently cationic: The term "permanently cationic" as
used herein will be recognized and understood by the person of
ordinary skill in the art, and means, for example, that the
respective compound, or group or atom, is positively charged at any
pH value or hydrogen ion activity of its environment. Typically,
the positive charge is results from the presence of a quaternary
nitrogen atom. Where a compound carries a plurality of such
positive charges, it may be referred to as permanently
polycationic, which is a subcategory of permanently cationic.
[0058] Pharmaceutically effective amount: The terms
"pharmaceutically effective amount" or "effective amount" will be
recognized and understood by the person of ordinary skill in the
art, and are for example intended to refer to an amount of a
compound (e.g. the artificial RNA of the invention) that is
sufficient to induce a pharmaceutical effect, such as, in the
context of the invention, an immune response (e.g. against an
antigenic peptide, protein, polyprotein as defined herein).
[0059] Polyvalent/multivalent vaccine, polyvalent/multivalent
composition: The terms "polyvalent vaccine", "polyvalent
composition" "multivalent vaccine" or "multivalent composition"
will be recognized and understood by the person of ordinary skill
in the art, and are for example intended to refer to a composition
or a vaccine comprising antigens from more than one strain of a
virus, or comprising different antigens of the same virus, or any
combination thereof. The terms describe that said vaccine or
composition has more than one valence. In the context of the
invention, a polyvalent RSV vaccine would comprise a vaccine
comprising an artificial RNA encoding antigenic peptides or
proteins derived from several different RSV strains or comprising
artificial RNA encoding different antigens from the same RSV
strain, or a combination thereof. In preferred embodiment, a
polyvalent RSV vaccine or composition comprises more than one,
preferably 2, 3, 4 or even more different artificial RNA species
each encoding at least one different antigenic peptide or protein
of RSV (e.g. RSV F and RSV M or RSV F and RSV N). Methods to
produce polyvalent mRNA vaccines are disclosed in the PCT
application PCT/EP2016/082487 or in published patent application
WO2017/1090134A1.
[0060] Stabilized nucleic acid molecule" or "stabilized RNA: The
term "stabilized nucleic acid molecule" or "stabilized RNA" refer
to a nucleic acid molecule, preferably an RNA molecule that is
modified such, that it is more stable to disintegration or
degradation, e.g., by environmental factors or enzymatic digest,
such as by an exo- or endonuclease degradation, than the nucleic
acid molecule without the modification. Preferably, a stabilized
nucleic acid molecule, e.g. stabilized RNA, in the context of the
present invention is stabilized in a cell, such as a prokaryotic or
eukaryotic cell, preferably in a mammalian cell, such as a human
cell. The stabilization effect may also be exerted outside of
cells, e.g. in a buffer solution etc., for example, in a
manufacturing process for a pharmaceutical composition comprising
the stabilized nucleic acid molecule.
[0061] T-cell responses: The terms "cellular immunity" or "cellular
immune response" or "cellular T-cell responses" as used herein will
be recognized and understood by the person of ordinary skill in the
art, and are for example intended to refer to the activation of
macrophages, natural killer cells (NK), antigen-specific cytotoxic
T-lymphocytes, and the release of various cytokines in response to
an antigen. In more general terms, cellular immunity is not based
on antibodies, but on the activation of cells of the immune system.
Typically, a cellular immune response may be characterized e.g. by
activating antigen-specific cytotoxic T-lymphocytes that are able
to induce apoptosis in cells, e.g. specific immune cells like
dendritic cells or other cells, displaying epitopes of foreign
antigens on their surface. In the context of the invention, the
antigen is provided by the artificial RNA encoding at least one
antigenic peptide or protein derived from RSV, suitably inducing
T-cell responses. The artificial RNA, the composition, the vaccine
of the invention advantageously elicit cellular T-cell responses
against RSV F antigens.
[0062] Variant (of a sequence): The term "variant" as used
throughout the present specification in the context of a nucleic
acid sequence will be recognized and understood by the person of
ordinary skill in the art, and is for example intended to refer to
a variant of nucleic acid sequences which forms the basis of a
nucleic acid sequence. For example, a variant nucleic acid sequence
may exhibit one or more nucleotide deletions, insertions, additions
and/or substitutions compared to the nucleic acid sequence from
which the variant is derived. Preferably, a variant of a nucleic
acid sequence is at least 40%, preferably at least 50%, more
preferably at least 60%, more preferably at least 70%, even more
preferably at least 80%, even more preferably at least 90%, most
preferably at least 95% identical to the nucleic acid sequence the
variant is derived from. Preferably, the variant is a functional
variant. A "variant" of a nucleic acid sequence may have at least
70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a
stretch of 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic
acid sequence.
[0063] The term "variant" as used throughout the present
specification in the context of proteins or peptides will be
recognized and understood by the person of ordinary skill in the
art, and is for example intended to refer to a proteins or peptide
variant having an amino acid sequence which differs from the
original sequence in one or more mutation(s), such as one or more
substituted, inserted and/or deleted amino acid(s). Preferably,
these fragments and/or variants have the same biological function
or specific activity compared to the full-length native protein,
e.g. its specific antigenic property. "Variants" of proteins or
peptides as defined in the context of the present invention may
comprise conservative amino acid substitution(s) compared to their
native, i.e. non-mutated physiological, sequence. Those amino acid
sequences as well as their encoding nucleotide sequences in
particular fall under the term variants as defined herein.
Substitutions in which amino acids, which originate from the same
class, are exchanged for one another are called conservative
substitutions. In particular, these are amino acids having
aliphatic side chains, positively or negatively charged side
chains, aromatic groups in the side chains or amino acids, the side
chains of which can enter into hydrogen bridges, e.g. side chains
which have a hydroxyl function. This means that e.g. an amino acid
having a polar side chain is replaced by another amino acid having
a likewise polar side chain, or, for example, an amino acid
characterized by a hydrophobic side chain is substituted by another
amino acid having a likewise hydrophobic side chain (e.g. serine
(threonine) by threonine (serine) or leucine (isoleucine) by
isoleucine (leucine)). Insertions and substitutions are possible,
in particular, at those sequence positions which cause no
modification to the three-dimensional structure or do not affect
the binding region. Modifications to a three-dimensional structure
by insertion(s) or deletion(s) can easily be determined e.g. using
CD spectra (circular dichroism spectra). A "variant" of a protein
or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or
99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100
amino acids of such protein or peptide. Preferably, a variant of a
protein comprises a functional variant of the protein, which means
that the variant exerts the same effect or functionality as the
protein it is derived from.
[0064] 3'-untranslated region, 3'-UTR element, 3'-UTR: The term
"3'-untranslated region" or "3'-UTR element" will be recognized and
understood by the person of ordinary skill in the art, and are for
example intended to refer to a part of a nucleic acid molecule,
which is located 3' (i.e. "downstream") of a coding sequence and
which is typically not translated into protein. Usually, a 3'-UTR
is the part of an mRNA which is located between the coding sequence
(cds) and the poly(A) sequence of the mRNA. In the context of the
invention, the term 3'-UTR may also comprise elements, which are
not encoded in the DNA template, from which an artificial RNA is
transcribed, but which are added after transcription during
maturation, e.g. a poly(A) sequence.
[0065] 5-untranslated region, 5'-UTR element, 5'-UTR: The term
"5'-untranslated region (5'-UTR)" will be recognized and understood
by the person of ordinary skill in the art, and are for example
intended to refer to a part of a nucleic acid molecule, which is
located 5' (i.e. "upstream") of a coding sequence and which is not
translated into protein. A 5'-UTR is typically understood to be a
particular section of messenger RNA (mRNA), which is located 5' of
the coding sequence of the mRNA. Typically, the 5'-UTR starts with
the transcriptional start site and ends one nucleotide before the
start codon of the coding sequence. Preferably, the 5'-UTRs have a
length of more than 20, 30, 40 or 50 nucleotides. The 5'-UTR may
comprise elements for controlling gene expression, also called
regulatory elements. Such regulatory elements may be, for example,
ribosomal binding sites. The 5'-UTR may be post-transcriptionally
modified, for example by addition of a 5'-cap.
[0066] 5-terminal oligopyrimidine tract (TOP), TOP-UTR: The term
"5'-terminal oligopyrimidine tract (TOP)" has to be understood as a
stretch of pyrimidine nucleotides located in the 5-terminal region
of a nucleic acid molecule, such as the 5-terminal region of
certain mRNA molecules or the 5-terminal region of a functional
entity, e.g. the transcribed region, of certain genes. The sequence
starts with a cytidine, which usually corresponds to the
transcriptional start site, and is followed by a stretch of usually
about 3 to 30 pyrimidine nucleotides. For example, the TOP may
comprise 3-30 or even more nucleotides. The pyrimidine stretch and
thus the 5'-TOP ends one nucleotide 5' to the first purine
nucleotide located downstream of the TOP. Messenger RNA that
contains a 5'-terminal oligopyrimidine tract is often referred to
as TOP mRNA. Accordingly, genes that provide such messenger RNAs
are referred to as TOP genes. The term "TOP motif" or "5'-TOP
motif" has to be understood as a nucleic acid sequence which
corresponds to a 5'-TOP as defined above. Thus, a TOP motif in the
context of the present invention is preferably a stretch of
pyrimidine nucleotides having a length of 3-30 nucleotides.
Preferably, the TOP-motif consists of at least 3 pyrimidine
nucleotides, preferably at least 4 pyrimidine nucleotides,
preferably at least 5 pyrimidine nucleotides, more preferably at
least 6 nucleotides, more preferably at least 7 nucleotides, most
preferably at least 8 pyrimidine nucleotides, wherein the stretch
of pyrimidine nucleotides preferably starts at its 5'-end with a
cytosine nucleotide. In TOP genes and TOP mRNAs, the TOP-motif
preferably starts at its 5'-end with the transcriptional start site
and ends one nucleotide 5' to the first purine residue in said gene
or mRNA. A TOP motif in the sense of the present invention is
preferably located at the 5'-end of a sequence which represents a
5'-UTR or at the 5'-end of a sequence which codes for a 5'-UTR.
Thus, preferably, a stretch of 3 or more pyrimidine nucleotides is
called "TOP motif" in the sense of the present invention if this
stretch is located at the 5'-end of a respective sequence, such as
the artificial nucleic acid, the 5'-UTR element of the artificial
nucleic acid, or the nucleic acid sequence which is derived from
the 5'-UTR of a TOP gene as described herein. In other words, a
stretch of 3 or more pyrimidine nucleotides, which is not located
at the 5'-end of a 5'-UTR or a 5'-UTR element but anywhere within a
5'-UTR or a 5'-UTR element, is preferably not referred to as "TOP
motif". In some embodiments, the nucleic acid sequence of the
5'-UTR element, which is derived from a 5'-UTR of a TOP gene,
terminates at its 3'-end with a nucleotide located at position 1,
2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (e.g.
A(U/T)G) of the gene or RNA it is derived from. Thus, the 5'-UTR
element does not comprise any part of the protein coding sequence.
Thus, preferably, the only protein coding part of the at least one
nucleic acid sequence, particularly of the RNA sequence, is
provided by the coding sequence.
SHORT DESCRIPTION OF THE INVENTION
[0067] The present invention is based on the inventor's surprising
finding that at least one peptide or protein derived from of a
Respiratory syncytial virus (RSV) F protein encoded by the
artificial RNA of the invention can efficiently be expressed in a
mammalian cell. Even more unexpected, the inventors showed that the
artificial RNA of the invention can induce specific functional and
protective immune responses in e.g. cotton rats (see e.g. Example
2, 3). Through different optimizations in RSV F antigen design, the
immune responses could be further improved. In addition, the
expression of the RSV F antigen encoded by the artificial nucleic
RNA could be increased by selecting suitable heterologous 5'
untranslated regions (UTRs) and suitable heterologous 3
untranslated regions (UTRs) (see e.g. Example 4). Advantageously,
said artificial RNA of the invention comprising advantageous
3-UTR/5'-UTR combinations induce very efficient antigen-specific
immune responses against the encoded RSV F. Further, artificial RNA
of the invention comprised in lipid nanoparticles (LNPs) very
efficiently induces antigen-specific immune responses against RSV F
at a very low dosages and dosing regimen (see e.g. Example 3).
Further, e.g. Example 8 and Example 12 provide
compositions/vaccines comprising a further artificial RNA encoding
a further antigen wherein said artificial RNA encoding a further
antigen suitably elicits or enhances T-cell responses and results
in a Th1-biased immune response, which is considered to be an
important prerequisite for a potential RSV vaccine (Th2-biased
responses have been associated with enhanced respiratory disease
(ERD) in animal models). Furthermore the compositions are suitable
to induce T-cell responses. Accordingly, the artificial RNA, and
the composition/vaccine comprising said artificial RNA of the
invention are suitable for eliciting an immune response against RSV
F in a mammalian subject. The artificial RNA and the
composition/vaccine comprising said artificial RNA is therefore
suitable for use as a vaccine, e.g. as a human vaccine, e.g. as a
vaccine for pregnant women or infants.
[0068] In a first aspect, the present invention provides an
artificial nucleic acid, preferably an artificial RNA comprising at
least one 5' untranslated region (UTR) and/or at least one 3'
untranslated region (UTR), and at least one coding sequence
operably linked to said 3'-UTR and/or 5'-UTR encoding at least one
antigenic peptide or protein derived from RSV F protein or a
fragment or variant thereof.
[0069] In preferred embodiments, the artificial RNA comprises at
least one nucleic acid sequence derived from a 3'-UTR of a gene
selected from an ALB7 gene, an alpha-globin gene, a PSMB3, CASP1,
COX6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or a
variant of any one of these genes
[0070] In preferred embodiments, the artificial RNA comprises at
least one nucleic acid sequence derived from a 5'-UTR of gene
selected from a RPL32 gene, a HSD17B4, ASAH1, ATP5A1, MP68, NDUFA4,
NOSIP, RPL31, SLC7A3, TUBB4 and UBQLN2, or from a homolog, a
fragment or variant of any one of these genes.
[0071] Suitably, the artificial RNA of the invention comprises at
least one coding sequence encoding at least one antigenic peptide
or protein derived from a RSV F protein operably linked to a 3'-UTR
and a 5'-UTR selected from a-1 (HSD17B4/PSMB3), a-2 (Ndufa4/PSMB3),
a-3 (SLC7A3/PSMB3), a-4 (NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1
(UBQLN2/RPS9), b-2 (ASAH1/RPS9), b-3 (HSD17B4/RPS9), b-4
(HSD17B4/CASP1), b-5 (NOSIP/COX6B1), c-1 (NDUFA4/RPS9), c-2
(NOSIP/NDUFA1), c-3 (NDUFA4/COX6B1), c-4 (NDUFA4/NDUFA1), c-5
(ATP5A1/PSMB3), d-1 (Rpl31/PSMB3), d-2 (ATP5A1/CASP1), d-3
(SLC7A3/GNAS), d-4 (HSD17B4/NDUFA1), d-5 (Slc7a3/Ndufa1), e-1
(TUBB4B/RPS9), e-2 (RPL31/RPS9), e-3 (MP68/RPS9), e-4 (NOSIP/RPS9),
e-5 (ATP5A1/RPS9), e-6 (ATP5A1/COX6B1), f-1 (ATP5A1/GNAS), f-2
(ATP5A1/NDUFA1), f-3 (HSD17B4/COX6B1), f-4 (HSD17B4/GNAS), f-5
(MP68/COX6B1), g-1 (MP68/NDUFA1), g-2 (NDUFA4/CASP1), g-3
(NDUFA4/GNAS), g-4 (NOSIP/CASP1), g-5 (RPL31/CASP1), h-1
(RPL31/COX6B1), h-2 (RPL31/GNAS), h-3 (RPL31/NDUFA1), h-4
(Slc7a3/CASP1), h-5 (SLC7A3/COX6B1), i-1 (SLC7A3/RPS9), i-2
(RPL32/ALB7), or i-3 (a-globin gene), wherein a-1 (HSD17B4/PSMB3),
a-4 (NDUFA4/PSMB3), c-1 (NDUFA4/RPS9), e-4 NOSIP/RPS9), g-2
(NDUFA4/CASP1), i-2 (RPL32/ALB7), or i-3 (alpha-globin) are
particularly preferred.
[0072] The at least one antigenic peptide or protein derived from
RSV F protein may be a full-length F protein (referred to as "F0",
aa 1-574) or an F protein with deleted C-terminus (referred to as
"F-del", aa 1-553), or a fragment or a variant thereof.
[0073] The at least one antigenic peptide or protein may
additionally comprise a mutation that stabilizes the antigen in
pre-conformation state/pre-fusion conformation, preferably a DSCav1
mutation (S155C, S290C, S190F, and V207L) or a fragment or a
variant, or a functional variant thereof (referred to as "DSCav1",
e.g. "F0_DSCav1" or "F-del_DSCav1").
[0074] The at least one antigenic peptide or protein may be a
fusion protein comprising the two subunits, F1 and F2 of mature F
into a single chain, connected via a linker (GS) to enhance
stability of the protein (F(1-103)-GS-F(145-574);
F(1-103)-GS-F(145-553)).
[0075] The protein comprising the two subunits of mature F into a
single chain (referred to as "F2-linker-F1"), e.g.
(F(1-103)-GS-F(145-574); F(1-103)-GS-F(145-553) may additionally
comprise a DSCav1 mutation (herein referred to as "mut0").
[0076] The protein comprising the two subunits of mature F into a
single chain (referred to as "F2-linker-F1") may additionally to
the DSCav1 mutation comprise at least one further mutation that
promotes inter-protomer disulphide bonds, wherein the mutations may
be selected from (S46G, A149C, S215P, Y458C, K465Q; herein referred
to as "mut1"), (S46G, E92D, A149C, S215P, Y458C, K465Q; herein
referred to as "mut2"), or (S46G, N671, E92D, A149C, S215P, Y458C,
K465Q; herein referred to as "mut3"), (A149C, Y458C; herein
referred to as "mut4"), (N183GC, N428C; herein referred to as
"mut5"), (Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M,
R429K, K465Q; herein referred to as "mut6"), (Q98C, Q361C, L95M,
I221M, R429K; herein referred to as "mut7"), or (N183GC, N428C,
S46G, N671, E92D, S215P, K465Q; herein referred to as "mut8") or a
fragment or a variant, or a functional variant thereof.
[0077] The at least one antigenic peptide or protein derived from
RSV F protein encoded by the artificial RNA of the invention may be
selected from F0, F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2,
F_DSCav1_mut3, F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6,
F_DSCav1_mut7, F_DSCav1_mut8, F_DSCav1_mut0, F-del_DSCav1,
F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3,
F-del_DSCav1_mut4, F-del_DSCav1_mut5, F-del_DSCav1_mut6,
F-del_DSCav1_mut7, F-del_DSCav1_mut8, F-del_DSCav1_mut0 are
preferred.
[0078] The at least one coding sequence may encode at least one of
the amino acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636,
2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695,
6064, 6433, 6802, 7171, 7540, 7909, 8279-9683, 11726, 12095, 12464,
12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571,
16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106,
19475 or a fragment or variant of any of these sequences.
[0079] Preferably, the artificial RNA may comprise a coding
sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to any one of SEQ ID NOs: 69-77, 484-492, 899-906, 1268-1275,
1637-1644, 2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489,
3851-3858, 4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703,
6065-6072, 6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917,
21363-21384, 11727-11734, 12096-12103, 12465-12472, 12834-12841,
13941-13948, 14310-14317, 14679-14686, 15048-15055, 15417-15424,
15786-15793, 13203-13210, 13572-13579, 16155-16162, 16524-16531,
16893-16900, 17262-17269, 17631-17638, 18000-18007, 18369-18376,
18738-18745, 19107-19114, 19476-19483. 21389-21410 or a fragment or
variant of any of these sequences.
[0080] The artificial RNA may comprise a codon modified coding
sequence selected from C maximized coding sequence, CAI maximized
coding sequence, human codon usage adapted coding sequence, G/C
content modified coding sequence, and G/C optimized coding
sequence, or any combination thereof.
[0081] The artificial RNA may be an mRNA, a viral RNA,
self-replicating RNA, a circular RNA, or a replicon RNA. In
preferred embodiments, the artificial RNA is an mRNA.
[0082] The artificial RNA, preferably mRNA, may further comprise at
least one selected from a cap structure, a poly(A)sequence, a
poly(C)sequence, a histone-stem loop, and/or a 3'-terminal sequence
element.
[0083] The artificial RNA of the invention preferably comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 78-482, 11735-12094,
21415-21417, 21561-21563, 21489, 21490, 21635, 21636 or a fragment
or variant of any of these (encoding F0), SEQ ID NOs: 493-897,
12104-12463, 21418-21420, 21564-21566, 21491, 21492, 21637, 21638
or a fragment or variant of any of these (encoding F-del), SEQ ID
NOs: 907-1266, 12473-12832, 21421-21423, 21567-21569, 21493-21495,
21639-21641 or a fragment or variant of any of these (encoding
F0_DSCav1), SEQ ID NOs: 1276-1635, 8278, 12842-13201, 21424-21426,
21570-21572, 21496-21498, 21642-21644 or a fragment or variant of
any of these (encoding F-del_DSCav1), SEQ ID NOs: 1645-2004,
13949-14308, 21433-21435, 21579-21581, 21505-21507, 21651-21653 or
a fragment or variant of any of these (encoding F_DSCav1_mut1), SEQ
ID NOs: 2014-2373, 14318-14677, 21436-21438, 21582-21584,
21508-21510, 21654-21656 or a fragment or variant of any of these
(encoding F-del_DSCav1_mut1), SEQ ID NOs: 2383-2742, 14687-15046,
21439-21441, 21585-21587, 21511-21513, 21657-21659 or a fragment or
variant of any of these (encoding F_DSCav1_mut2), SEQ ID NOs:
2752-3111, 15056-15415, 21442-21444, 21588-21590, 21514-21516,
21660-21662 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut2), SEQ ID NOs: 3121-3480, 15425-15784,
21445-21447, 21591-21593, 21517-21519, 21663-21665 or a fragment or
variant of any of these (encoding F_DSCav1_mut3), SEQ ID NOs:
3490-3849, 15794-16153, 21448-21450, 21594-21596, 21520-21522,
21666-21668 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut3), SEQ ID NOs: 3859-4218, 13211-13570,
21427-21429, 21573-21575, 21499-21501, 21645-21647 or a fragment or
variant of any of these (encoding F_DSCav1_mut0), SEQ ID NOs:
4228-4587, 13580-13939, 21430-21432, 21576-21578, 21502-21504,
21648-21650 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut0), SEQ ID NOs: 4597-4956, 16163-16522,
21451-21453, 21597-21599, 21523-21525, 21669-21671 or a fragment or
variant of any of these (encoding F_DSCav1_mut4), SEQ ID NOs:
4966-5325, 16532-16891, 21454-21456, 21600-21602, 21526-21528,
21672-21674 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut4), SEQ ID NOs: 5335-5694, 16901-17260,
21457-21459, 21603-21605, 21529-21531, 21675-21677 or a fragment or
variant of any of these (encoding F_DSCav1_mut5), SEQ ID NOs:
5704-6063, 17270-17629, 21460-21462, 21606-21608, 21532-21534,
21678-21680 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut5), SEQ ID NOs: 6073-6432, 17639-17998,
21463-21465, 21609-21611, 21535-21537, 21681-21683 or a fragment or
variant of any of these (encoding F_DSCav1_mut6), SEQ ID NOs:
6442-6801, 18008-18367, 21466-21468, 21612-21614, 21538-21540,
21684-21686 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut6), SEQ ID NOs: 6811-7170, 18377-18736,
21469-21471, 21615-21617, 21541-21543, 21687-21689 or a fragment or
variant of any of these (encoding F_DSCav1_mut7), SEQ ID NOs:
7180-7539, 18746-19105, 21472-21474, 21618-21620, 21544-21546,
21690-21692 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut7), SEQ ID NOs: 7549-7908, 19115-19474,
21475-21477, 21621-21623, 21547-21549, 21693-21695 or a fragment or
variant of any of these (encoding F_DSCav1_mut8), or SEQ ID NOs:
7918-8277, 19484-19843, 21478-21480, 21624-21626, 21550-21552,
21696-21698 or a fragment or variant of any of these (encoding
F-del_DSCav1_mut8).
[0084] In a second aspect, the present invention provides a
composition comprising the artificial RNA of the first aspect.
[0085] In preferred embodiments, the composition comprising the
artificial RNA of the first aspect comprises at least one further
artificial RNA comprising at least one coding sequence encoding at
least one antigenic peptide or protein derived from RSV selected
from matrix protein M, nucleoprotein N, M2-1 protein, and/or
phosphoprotein P or combinations thereof.
[0086] Matrix protein M, nucleoprotein N, M2-1 protein, M2-2
protein, and/or phosphoprotein P are suitable T-cell antigens and
may promote efficient T-cell responses of the composition or
vaccine when administered to a subject.
[0087] The further artificial RNA may comprise a coding sequence
being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one
of SEQ ID NOs: 9685-9692, 10135-10142, 10638-10645, 11184-11191,
21385-21388, 19845-19852, 20214-20221, 20583-20590, 20952-20959,
21411-21414 or a fragment or variant of any of these sequences.
[0088] Suitably, said further artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from SEQ ID NOs:
9693-10052, 10143-10502, 10646-11005, 11192-11551, 19853-20212,
20222-20581, 20591-20950, 20960-21319, 21481-21488, 21627-21634,
21553-21560, 21699-21706 or a fragment or variant of any of these
sequences.
[0089] Suitably, the composition may comprise the artificial RNA of
the invention complexed with, encapsulated in, or associated with
one or more lipids, thereby forming lipid nanoparticles.
[0090] The composition may preferably comprise the artificial RNA
of the invention complexed with one or more lipids thereby forming
lipid nanoparticles (LNP), wherein the LNP essentially consists
of
[0091] (i) at least one cationic lipid as defined herein,
preferably a lipid of formula (III), more preferably lipid
III-3;
[0092] (ii) a neutral lipid as defined herein, preferably
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);
[0093] (iii) a steroid or steroid analogue as defined herein,
preferably cholesterol; and
[0094] (iv) a PEG-lipid as defined herein, e.g. PEG-DMG or
PEG-cDMA, preferably a PEGylated lipid of formula (IVa); wherein
(i) to (iv) are in a molar ratio of about 20-60% cationic lipid:
5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.
[0095] The present invention also concerns a RSV vaccine comprising
said artificial RNA or said composition.
[0096] The present invention is also directed to the use of the
artificial RNA, the composition and the vaccine in treatment or
prophylaxis of an infection with RSV.
[0097] In particular, the present invention is directed to the use
of the artificial RNA, the composition and the vaccine in treatment
or prophylaxis of an infection with RSV or a disorder related to
such an infection.
[0098] The invention further concerns a method of treating or
preventing a disorder or a disease in a subject, first and second
medical uses of the artificial RNA, compositions and vaccines.
Further, the invention is directed to a kit, particularly to a kit
of parts, comprising the artificial RNA, compositions and
vaccines.
DETAILED DESCRIPTION OF THE INVENTION
[0099] The present application is filed together with a sequence
listing in electronic format, which is part of the description of
the present application (WIPO standard ST.25). The information
contained in the electronic format of the sequence listing filed
together with this application is incorporated herein by reference
in its entirety. Where reference is made herein to a "SEQ ID NO"
the corresponding nucleic acid sequence or amino acid (aa) sequence
in the sequence listing having the respective identifier is
referred to. For many sequences, the sequence listing also provides
additional detailed information, e.g. regarding certain structural
features, sequence optimizations, GenBank identifiers, or
additional detailed information regarding its coding capacity. In
particular, such information is provided under numeric identifier
<223> in the WIPO standard ST.25 sequence listing.
Accordingly, information provided under said numeric identifier
<223> is explicitly included herein in its entirety and has
to be understood as integral part of the description of the
underlying invention.
Artificial Nucleic Acid:
[0100] In a first aspect, the invention relates to an artificial
nucleic acid comprising [0101] a) at least one heterologous 5'
untranslated region (5'-UTR) and/or at least one heterologous 3'
untranslated region (3'-UTR); and [0102] b) at least one coding
sequence operably linked to said 3'-UTR and/or 5'-UTR encoding at
least one antigenic peptide or protein derived from a Respiratory
syncytial virus (RSV) or a fragment or variant thereof.
[0103] In a preferred embodiment of the first aspect, the invention
relates to an artificial RNA, preferably an RNA suitable for
vaccination, comprising [0104] a) at least one heterologous 5'
untranslated region (5'-UTR) and/or at least one heterologous 3'
untranslated region (3'-UTR); and [0105] b) at least one coding
sequence operably linked to said 3'-UTR and/or 5'-UTR encoding at
least one antigenic peptide or protein derived from a RSV fusion
(F) protein or a fragment or variant thereof.
[0106] In general, the RNA of the invention may be composed of a
protein-coding region, and 5'- and/or 3-untranslated regions
(UTRs). The 3'-UTR is variable in sequence and size; it spans
between the stop codon and the poly(A) tail. Importantly, the
3'-UTR sequence harbors several regulatory motifs that determine
RNA turnover, stability and localization, and thus governs many
aspects of post-transcriptional regulation. In medical application
of RNA (e.g. immunotherapy applications, vaccination) the
regulation of RNA translation into protein is of paramount
importance to therapeutic safety and efficacy. The present
inventors surprisingly discovered that certain combinations of
3-UTRs and/or 5'-UTRs act in concert to synergistically enhance the
expression of operably linked nucleic acid sequences encoding RSV
antigenic peptides or proteins. Artificial RNA molecules harboring
the inventive UTR combinations advantageously enable the rapid and
transient expression of high amounts of RSV antigenic peptides or
proteins derived from RSV F. Accordingly, the artificial RNA
provided herein is particularly useful and suitable for various
applications in vivo, including the vaccination against RSV.
[0107] Suitably, the artificial RNA may comprise at least one
heterologous 5'-UTR and/or at least one heterologous 3'-UTR. In
this context, an UTR of the invention comprises or consists of a
nucleic acid sequence derived from a 5'-UTR or a 3'-UTR of any
naturally occurring gene or a fragment, a homolog or a variant
thereof. Preferably, a 5-UTR or a 3'-UTR of the invention is
heterologous to the at least one coding sequence encoding the at
least one antigenic peptide or protein derived from RSV F. Suitable
heterologous 5'-UTRs or heterologous 3'-UTRs are derived from
naturally occurring genes (that are not derived from RSV). In other
embodiments, synthetically engineered 5'-UTRs or 3-UTRs may be used
in the context of the present invention.
[0108] In preferred embodiments, the at least one artificial RNA
comprises at least one heterologous 3'-UTR.
[0109] Preferably, the at least one heterologous 3'-UTR comprises
or consists of a nucleic acid sequence derived from a 3'-UTR of a
chordate gene, preferably a vertebrate gene, more preferably a
mammalian gene, most preferably a human gene, or from a variant of
a 3'-UTR of a chordate gene, preferably a vertebrate gene, more
preferably a mammalian gene, most preferably a human gene.
[0110] Preferably the artificial RNA of the present invention
comprises a 3'-UTR, which may be derivable from a gene that relates
to an RNA with an enhanced half-life (that provides a stable RNA),
for example a 3'-UTR as defined and described below.
[0111] Preferably, the at least one heterologous 3'-UTR comprises a
nucleic acid sequence derived from a 3'-UTR of a gene, which
preferably encodes a stable mRNA, or from a homolog, a fragment or
a variant of said gene.
[0112] In preferred embodiments of the first aspect, the artificial
RNA of the invention comprises at least one heterologous 3'-UTR,
wherein the at least one heterologous 3'-UTR comprises a nucleic
acid sequence derived from a 3'-UTR of a gene selected from PSMB3,
ALB7, alpha-globin (referred to as "muag"), CASP1, COX6B1, GNAS,
NDUFA1 and RPS9, or from a homolog, a fragment or variant of any
one of these genes.
[0113] ALB7-derived 3'-UTR: In preferred embodiments, the 3'-UTR
comprises or consists of a nucleic acid sequence which is derived
from the 3'-UTR of a vertebrate albumin gene or from a variant
thereof, preferably from the 3'-UTR of a mammalian albumin gene or
from a variant thereof, more preferably from the 3'-UTR of a human
albumin gene or from a variant thereof, even more preferably from
the 3'-UTR of the human albumin gene according to GenBank Accession
number NM_000477.5, or from a homolog, fragment or variant thereof.
Accordingly, the artificial RNA of the invention may comprise a
3'-UTR derived from a ALB7 gene, wherein said 3'-UTR derived from a
ALB7 gene comprises or consists of a nucleic acid sequence being
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:
35 or 36 or a fragment or a variant thereof.
[0114] Alpha-globin gene-derived 3'-UTR: In preferred embodiments,
the 3'-UTR comprises or consists of a nucleic acid sequence which
is derived from the 3'-UTR of a vertebrate alpha-globin gene
(referred to as "muag") or from a variant thereof, preferably from
the 3'-UTR of a mammalian alpha-globin or from a variant thereof,
more preferably from the 3'-UTR of a human alpha-globin gene or
from a variant thereof, even more preferably from the 3'-UTR of the
human alpha-globin gene. Accordingly, the RNA of the invention may
comprise a 3'-UTR derived from a alpha-globin gene, wherein said
3'-UTR derived from a alpha-globin gene comprises or consists of a
nucleic acid sequence being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NO: 37 or 38 or a fragment or a variant
thereof.
[0115] PSMB3-derived 3'-UTR: The artificial RNA of the invention
may comprise a 3'-UTR which is derived from a 3'-UTR of a gene
encoding a proteasome subunit beta type-3 (PSMB3) protein, or a
homolog, variant, fragment or derivative thereof. Such 3'-UTRs
preferably comprise or consist of a nucleic acid sequences derived
from the 3'-UTR of a proteasome subunit beta type-3 (PSMB3) gene,
preferably from a vertebrate, more preferably a mammalian, most
preferably a human proteasome subunit beta type-3 (PSMB3) gene, or
a homolog, variant, fragment or derivative thereof. Said gene may
preferably encode a proteasome subunit beta type-3 (PSMB3) protein
corresponding to a human proteasome subunit beta type-3 (PSMB3)
protein (UniProt Ref. No. P49720, entry version #183 of 30 Aug.
2017). Accordingly, the artificial RNA of the invention may
comprise a 3'-UTR derived from a PSMB3 gene, wherein said 3'-UTR
derived from a PSMB3 gene comprises or consists of a nucleic acid
sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO: 23 or 24 or a fragment or a variant thereof.
[0116] CASP1-derived 3'-UTR: The artificial RNA of the invention
may comprise a 3'-UTR which is derived from a 3'-UTR of a gene
encoding a Caspase-1 (CASP1) protein, or a homolog, variant,
fragment or derivative thereof. Such 3'-UTRs preferably comprise or
consist of a nucleic acid sequence derived from the 3'-UTR of a
Caspase-1 (CASP1) gene, preferably from a vertebrate, more
preferably a mammalian, most preferably a human Caspase-1 (CASP1)
gene, or a homolog, variant, fragment or derivative thereof.
Accordingly, the RNA of the invention may comprise a 3'-UTR derived
from a CASP1 gene, wherein said 3'-UTR derived from a CASP1 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25 or 26 or
a fragment or a variant thereof.
[0117] COX6B1-derived 3'-UTR: The artificial RNA of the invention
may comprise a 3'-UTR which is derived from a 3'-UTR of a COX6B1
gene encoding a cytochrome c oxidase subunit 6B1 (COX6B1) protein,
or a homolog, variant, fragment or derivative thereof. Such 3'-UTRs
preferably comprise or consist of a nucleic acid sequence which is
derived from the 3'-UTR of a cytochrome c oxidase subunit 6B1
(COX6B1) gene, preferably from a vertebrate, more preferably a
mammalian, most preferably a human cytochrome c oxidase subunit 6B1
(COX6B1) gene, or a homolog, variant, fragment or derivative
thereof. Said gene may preferably encode a cytochrome c oxidase
subunit 681 (COX6B1) protein corresponding to a human cytochrome c
oxidase subunit 6B1 (COX6B1) protein (UniProt Ref. No. P14854,
entry version #166 of 30 Aug. 2017). Accordingly, the artificial
RNA of the invention may comprise a 3'-UTR derived from a COX6B1
gene, wherein said 3'-UTR derived from a COX6B1 gene comprises or
consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 27 or 28 or a
fragment or a variant thereof.
[0118] GNAS-derived 3'-UTR: The artificial RNA of the invention may
comprise a 3'-UTR which is derived from a 3'-UTR of a GNAS gene
encoding a Guanine nucleotide-binding protein G(s) subunit alpha
isoforms short (GNAS) protein, or a homolog, variant, fragment or
derivative thereof. Such 3-UTRs preferably comprise or consist of a
nucleic acid sequence which is derived from the 3'-UTR of a Guanine
nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS)
gene, preferably from a vertebrate, more preferably a mammalian
Guanine nucleotide-binding protein G(s) subunit alpha isoforms
short (GNAS) gene, or a homolog, variant, fragment or derivative
thereof. Said gene may preferably encode a Guanine
nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS)
protein corresponding to a human Guanine nucleotide-binding protein
G(s) subunit alpha isoforms short (GNAS) protein (UniProt Ref. No.
P63092, entry version #153 of 30 Aug. 2017). Accordingly, the
artificial RNA of the invention may comprise a 3'-UTR derived from
a GNAS gene, wherein said 3'-UTR derived from a GNAS gene comprises
or consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 30 or a
fragment or a variant thereof.
[0119] NDUFA1-derived 3'-UTR: The artificial RNA of the invention
may comprise a 3'-UTR which is derived from a 3'-UTR of a gene
encoding a NADH dehydrogenase [ubiquinone] 1 alpha sub complex
subunit 1 (NDUFA1) protein, or a homolog, variant, fragment or
derivative thereof. Such 3'-UTRs preferably comprise or consist of
a nucleic acid sequence derived from the 3'-UTR of a NADH
dehydrogenase [ubiquinone] 1 alpha sub complex subunit 1 (NDUFA1)
gene, preferably from a vertebrate, more preferably a mammalian
NADH dehydrogenase [ubiquinone]1 alpha sub complex subunit 1
(NDUFA1) gene, or a homolog, variant, fragment or derivative
thereof. Said gene may preferably encode a NADH dehydrogenase
[ubiquinone] 1 alpha sub complex subunit 1 (NDUFA1) protein
corresponding to a human NADH dehydrogenase [ubiquinone] 1 alpha
sub complex subunit 1 (NDUFA1) protein (UniProt Ref. No. 015239,
entry version #152 of 30 Aug. 2017). Accordingly, the artificial
RNA of the invention may comprise a 3'-UTR derived from a NDUFA1
gene, wherein said 3'-UTR derived from a NDUFA1 gene comprises or
consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 31 or 32 or a
fragment or a variant thereof.
[0120] RPS9-derived 3'-UTR: The artificial RNA of the invention may
comprise a 3'-UTR which is derived from a 3'-UTR of a gene encoding
a 40S ribosomal protein S9 (RPS9) protein, or a homolog, variant,
fragment or derivative thereof. Such 3'-UTRs preferably comprise or
consist of a nucleic acid sequence derived from the 3'-UTR of a 40S
ribosomal protein S9 (RPS9) gene, preferably from a vertebrate,
more preferably a mammalian, most preferably a human 40S ribosomal
protein S9 (RPS9) gene, or a homolog, variant, fragment or
derivative thereof. Said gene may preferably encode a 40S ribosomal
protein S9 (RPS9) protein corresponding to a human 40S ribosomal
protein S9 (RPS9) protein (UniProt Ref. No. P46781, entry version
#179 of 30 Aug. 2017). Accordingly, the artificial RNA of the
invention may comprise a 3'-UTR derived from a RPS9 gene, wherein
said 3'-UTR derived from a RPS9 gene comprises or consists of a
nucleic acid sequence being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NO: 33 or 34 or a fragment or a variant
thereof.
[0121] Further 3'-UTRs: In embodiments, the artificial RNA as
defined herein comprises a 3'-UTR as described in WO2016/107877. In
this context, the disclosure of WO2016/107877 relating to 3'-UTR
sequences is herewith incorporated by reference. Particularly
suitable 3'-UTRs are SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to 318
of patent application WO2016/107877, or fragments or variants of
these sequences. Accordingly, the 3'-UTRs of the artificial RNA of
the present invention may comprise or consists of a corresponding
RNA sequence of the nucleic acid sequence according SEQ ID NOs: 1
to 24 and SEQ ID NOs: 49 to 318 of the patent application
WO2016/107877. In other embodiments, the artificial RNA as defined
herein comprises a 3'-UTR as described in WO2017/036580. In this
context, the disclosure of WO2017/036580 relating to 3'-UTR
sequences is herewith incorporated by reference. Particularly
suitable 3-UTRs are SEQ ID NOs: 152 to 204 of the patent
application WO2017/036580, or fragments or variants of these
sequences. Accordingly, the 3'-UTR of the artificial RNA of the
present invention may comprise or consist of a corresponding RNA
sequence of the nucleic acid sequence according SEQ ID NOs: 152 to
204 of the patent application WO2017/036580.
[0122] According to preferred embodiments the artificial RNA
comprises at least one heterologous 5'-UTR.
[0123] In preferred embodiments, the at least one artificial
nucleic acid as defined herein, particularly the RNA as defined
herein may comprise at least one heterologous 5'-UTR.
[0124] Preferably, the at least one 5'-UTR comprises or consists of
a nucleic acid sequence derived from the 5'-UTR of a chordate gene,
preferably a vertebrate gene, more preferably a mammalian gene,
most preferably a human gene, or from a variant of the 5'-UTR of a
chordate gene, preferably a vertebrate gene, more preferably a
mammalian gene, most preferably a human gene.
[0125] Preferably the artificial RNA of the present invention
comprises a 5'-UTR, which may be derivable from a gene that relates
to an RNA with an enhanced half-life (that provides a stable RNA),
for example a 5'-UTR as defined and described below.
[0126] Preferably, the at least one heterologous 5'-UTR comprises a
nucleic acid sequence derived from a 5'-UTR of a gene, which
preferably encodes a stable mRNA, or from a homolog, a fragment or
a variant of said gene.
[0127] In preferred embodiments of the first aspect, the artificial
RNA of the invention comprises at least one heterologous 5'-UTR,
wherein the at least one heterologous 5'-UTR comprises a nucleic
acid sequence derived from a 5'-UTR of gene selected from HSD17B4,
RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B,
and UBQLN2, or from a homolog, a fragment or variant of any one of
these genes.
[0128] RPL32-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR derived from a 5'-UTR of a gene encoding a
60S ribosomal protein L32, or a homolog, variant, fragment or
derivative thereof, wherein said 5'-UTR preferably lacks the 5'TOP
motif. Such 5'-UTRs preferably comprise or consist of a nucleic
acid sequence derived from the 5'-UTR of a 60S ribosomal protein
L32 (RPL32) gene, preferably from a vertebrate, more preferably a
mammalian, most preferably a human 60S ribosomal protein L32
(RPL32) gene, or a homolog, variant, fragment or derivative
thereof, wherein the 5'-UTR preferably does not comprise the 5'TOP
of said gene. Said gene may preferably encode a 60S ribosomal
protein L32 (RPL32) corresponding to a human 60S ribosomal protein
L32 (RPL32) (UniProt Ref. No. P62899, entry version #138 of 30 Aug.
2017). Accordingly, the artificial RNA of the invention may
comprise a 5'-UTR derived from a RPL32 gene, wherein said 5'-UTR
derived from a RPL32 gene comprises or consists of a nucleic acid
sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO: 21 or 22 or a fragment or a variant thereof.
[0129] HSD17B4-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR derived from a 5'-UTR of a gene encoding a
17-beta-hydroxysteroid dehydrogenase 4, or a homolog, variant,
fragment or derivative thereof, preferably lacking the 5'TOP motif.
Such 5'-UTRs preferably comprise or consist of a nucleic acid
sequence derived from the 5'-UTR of a 17-beta-hydroxysteroid
dehydrogenase 4 (also referred to as peroxisomal multifunctional
enzyme type 2) gene, preferably from a vertebrate, more preferably
mammalian, most preferably human 17-beta-hydroxysteroid
dehydrogenase 4 (HSD17B4) gene, or a homolog, variant, fragment or
derivative thereof, wherein preferably the 5'-UTR does not comprise
the 5'TOP of said gene. Said gene may preferably encode a
17-beta-hydroxysteroid dehydrogenase 4 protein corresponding to
human 17-beta-hydroxysteroid dehydrogenase 4 (UniProt Ref. No.
Q9BPX1, entry version #139 of Aug. 30, 2017), or a homolog,
variant, fragment or derivative thereof. Accordingly, the
artificial RNA of the invention may comprise a 5'-UTR derived from
a HSD17B4 gene, wherein said 5'-UTR derived from a HSD17B4 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or 2 or a
fragment or a variant thereof.
[0130] ASAH1-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR derived from a 5'-UTR of a gene encoding acid
ceramidase (ASAH1), or a homolog, variant, fragment or derivative
thereof. Such 5'-UTRs preferably comprise or consist of a nucleic
acid sequence derived from the 5'-UTR of an acid ceramidase (ASAH1)
gene, preferably from a vertebrate, more preferably mammalian, most
preferably human acid ceramidase (ASAH1) gene, or a homolog,
variant, fragment or derivative thereof. Said gene may preferably
encode an acid ceramidase (ASAH1) protein corresponding to human
acid ceramidase (ASAH1) (UniProt Ref. No. Q13510, entry version
#177 of Jun. 7, 2017), or a homolog, variant, fragment or
derivative thereof. Accordingly, the artificial RNA of the
invention may comprise a 5'-UTR derived from a ASAH1 gene, wherein
said 5'-UTR derived from a ASAH1 gene comprises or consists of a
nucleic acid sequence being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NO: 3 or 4 or a fragment or a variant
thereof.
[0131] ATP5A1-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR which is derived from a 5'-UTR of a gene
encoding mitochondrial ATP synthase subunit alpha (ATP5A1), or a
homolog, variant, fragment or derivative thereof, wherein said
5'-UTR preferably lacks the 5'TOP motif. Such 5'-UTRs preferably
comprise or consist of a nucleic acid sequence derived from the
5'-UTR of a mitochondrial ATP synthase subunit alpha (ATP5A1) gene,
preferably from a vertebrate, more preferably a mammalian and most
preferably a human mitochondrial ATP synthase subunit alpha
(ATP5A1) gene, or a homolog, variant, fragment or derivative
thereof, wherein the 5'-UTR preferably does not comprise the 5'TOP
of said gene. Said gene may preferably encode a mitochondrial ATP
synthase subunit alpha protein corresponding to human acid
mitochondrial ATP synthase subunit alpha (UniProt Ref. No. P25705,
entry version #208 of Aug. 30, 2017), or a homolog, variant,
fragment or derivative thereof. Accordingly, the artificial RNA of
the invention may comprise a 5'-UTR derived from a ATP5A1 gene,
wherein said 5'-UTR derived from a ATP5A1 gene comprises or
consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 5 or 6 or a fragment
or a variant thereof.
[0132] MP68-derived 5'-UTR: The artificial RNA of the invention may
comprise a 5'-UTR which is derived from a 5'-UTR of a gene encoding
MP68, or a homolog, fragment or variant thereof. Such 5'-UTRs
preferably comprise or consist of a nucleic acid sequence derived
from the 5'-UTR of a 6.8 kDa mitochondrial proteolipid (MP68) gene,
preferably from a vertebrate, more preferably a mammalian 6.8 kDa
mitochondrial proteolipid (MP68) gene, or a homolog, variant,
fragment or derivative thereof. Said gene may preferably encode a
6.8 kDa mitochondrial proteolipid (MP68) protein corresponding to a
human 6.8 kDa mitochondrial proteolipid (MP68) protein (UniProt
Ref. No. P56378, entry version #127 of 15 Feb. 2017). Accordingly,
the artificial RNA of the invention may comprise a 5'-UTR derived
from a MP68 gene, wherein said 5'-UTR derived from a MP68 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 8 or a
fragment or a variant thereof.
[0133] NDUFA4-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR which is derived from a 5'-UTR of a gene
encoding a Cytochrome c oxidase subunit (NDUFA4), or a homolog,
fragment or variant thereof. Such 5'-UTRs preferably comprise or
consist of a nucleic acid sequence derived from the 5'-UTR of a
Cytochrome c oxidase subunit (NDUFA4) gene, preferably from a
vertebrate, more preferably a mammalian Cytochrome c oxidase
subunit (NDUFA4) gene, or a homolog, variant, fragment or
derivative thereof. Said gene may preferably encode a Cytochrome c
oxidase subunit (NDUFA4) protein corresponding to a human
Cytochrome c oxidase subunit (NDUFA4) protein (UniProt Ref. No.
000483, entry version #149 of 30 Aug. 2017). Accordingly, the
artificial RNA of the invention may comprise a 5'-UTR derived from
a NDUFA4 gene, wherein said 5'-UTR derived from a NDUFA4 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 or 10 or
a fragment or a variant thereof.
[0134] NOSIP-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR which is derived from a 5-UTR of a gene
encoding a Nitric oxide synthase-interacting (NOSIP) protein, or a
homolog, variant, fragment or derivative thereof. Such 5'-UTRs
preferably comprise or consist of a nucleic acid sequence derived
from the 5'-UTR of a Nitric oxide synthase-interacting protein
(NOSIP) gene, preferably from a vertebrate, more preferably a
mammalian, most preferably a human Nitric oxide
synthase-interacting protein (NOSIP) gene, or a homolog, variant,
fragment or derivative thereof. Said gene may preferably encode a
Nitric oxide synthase-interacting protein (NOSIP) protein
corresponding to a human Nitric oxide synthase-interacting protein
(NOSIP) protein (UniProt Ref. No. Q9Y314, entry version #130 of 7
Jun. 2017). Accordingly, the artificial RNA of the invention may
comprise a 5'-UTR derived from a NOSIP gene, wherein said 5'-UTR
derived from a NOSIP gene comprises or consists of a nucleic acid
sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO: 11 or 12 or a fragment or a variant thereof.
[0135] RPL31-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR which is derived from a 5'-UTR of a gene
encoding a 60S ribosomal protein L31, or a homolog, variant,
fragment or derivative thereof, wherein said 5'-UTR preferably
lacks the 5'TOP motif. Such 5'-UTR preferably comprise or consist
of a nucleic acid sequence derived from the 5'-UTR of a 60S
ribosomal protein L31 (RPL31) gene, preferably from a vertebrate,
more preferably a mammalian60S ribosomal protein L31 (RPL31) gene,
or a homolog, variant, fragment or derivative thereof, wherein the
5'-UTR preferably does not comprise the 5'TOP of said gene. Said
gene may preferably encode a 60S ribosomal protein L31 (RPL31)
corresponding to a human 60S ribosomal protein L31 (RPL31) (UniProt
Ref. No. P62899, entry version #138 of 30 Aug. 2017). Accordingly,
the artificial RNA of the invention may comprise a 5'-UTR derived
from a RPL31 gene, wherein said 5'-UTR derived from a RPL31 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or 14 or
a fragment or a variant thereof.
[0136] SLC7A3-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR which is derived from a 5'-UTR of a gene
encoding a cationic amino acid transporter 3 (solute carrier family
7 member 3, SLC7A3) protein, or a homolog, variant, fragment or
derivative thereof. Such 5'-UTRs preferably comprise or consist of
a nucleic acid sequence derived from the 5'-UTR of a cationic amino
acid transporter 3 (SLC7A3) gene, preferably from a vertebrate,
more preferably a mammalian cationic amino acid transporter 3
(SLC7A3) gene, or a homolog, variant, fragment or derivative
thereof. Said gene may preferably encode a cationic amino acid
transporter 3 (SLC7A3) protein corresponding to a human cationic
amino acid transporter 3 (SLC7A3) protein (UniProt Ref. No. Q8WY07,
entry version #139 of 30 Aug. 2017). Accordingly, the artificial
RNA of the invention may comprise a 5'-UTR derived from a SLC7A3
gene, wherein said 5'-UTR derived from a SLC7A3 gene comprises or
consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 16 or a
fragment or a variant thereof.
[0137] TUBB4B-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR which is derived from a 5'-UTR of a gene
encoding a tubulin beta-4B chain (TUBB4B) protein, or a homolog,
variant, fragment or derivative thereof. Such 5'-UTRs preferably
comprise or consist of a nucleic acid sequence derived from the
5'-UTR of a tubulin beta-4B chain (TUBB4B) gene, preferably from a
vertebrate, more preferably a mammalian and most preferably a human
tubulin beta-48 chain (TUBB4B) gene, or a homolog, variant,
fragment or derivative thereof. Said gene may preferably encode a
tubulin beta-4B chain (TUBB4B) protein corresponding to human
tubulin beta-4B chain (TUBB4B) protein (UniProt Ref. No. Q8WY07,
entry version #142 of 30 Aug. 2017). Accordingly, the artificial
RNA of the invention may comprise a 5'-UTR derived from a TUBB4B
gene, wherein said 5'-UTR derived from a TUBB4B gene comprises or
consists of a nucleic acid sequence being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 or 18 or a
fragment or a variant thereof.
[0138] UBQLN2-derived 5'-UTR: The artificial RNA of the invention
may comprise a 5'-UTR which is derived from a 5'-UTR of a gene
encoding an ubiquilin-2 (UBQLN2) protein, or a homolog, variant,
fragment or derivative thereof. Such 5'-UTRs preferably comprise or
consist of a nucleic acid sequence derived from the 5'-UTR of an
ubiquilin-2 (UBQLN2) gene, preferably from a vertebrate, more
preferably a mammalian ubiquilin-2 (UBQLN2) gene, or a homolog,
variant, fragment or derivative thereof. Said gene may preferably
encode an ubiquilin-2 (UBQLN2) protein corresponding to UniProt
Ref. No. Q9UHD9, entry version #151 of 30 Aug. 2017. Accordingly,
the artificial RNA of the invention may comprise a 5'-UTR derived
from a UBQLN2 gene, wherein said 5'-UTR derived from a UBQLN2 gene
comprises or consists of a nucleic acid sequence being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19 or 20 or
a fragment or a variant thereof.
[0139] Further 5'-UTRs: In embodiments, the artificial RNA as
defined herein comprises a 5'-UTR as described in WO2013/143700. In
this context, the disclosure of WO2013/143700 relating to 5'-UTR
sequences is herewith incorporated by reference. Particularly
preferred 5'-UTRs are nucleic acid sequences derived from SEQ ID
NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422
of the patent application WO2013/143700, or fragments or variants
of these sequences. In this context, it is preferred that the
5'-UTR of the artificial RNA according to the present invention
comprises or consists of a corresponding RNA sequence of the
nucleic acid sequence according SEQ ID NOs: 1-1363, SEQ ID NO:
1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application
WO2013/143700. In other embodiments, the artificial RNA of the
invention comprises a 5'-UTR as described in WO2016/107877. In this
context, the disclosure of WO2016/107877 relating to 5'-UTR
sequences is herewith incorporated by reference. Particularly
preferred 5'-UTRs are nucleic acid sequences according to SEQ ID
NOs: 25 to 30 and SEQ ID NOs: 319 to 382 of the patent application
WO2016/107877, or fragments or variants of these sequences. In this
context, it is particularly preferred that the 5'-UTR of the
artificial RNA comprises or consists of a corresponding RNA
sequence of the nucleic acid sequence according SEQ ID NOs: 25 to
30 and SEQ ID NOs: 319 to 382 of the patent application
WO2016/107877. In other embodiments, the artificial RNA of the
invention comprises a 5'-UTR as described in WO2017/036580. In this
context, the disclosure of WO2017/036580 relating to 5'-UTR
sequences is herewith incorporated by reference. Particularly
preferred 5'-UTRs are nucleic acid sequences according to SEQ ID
NOs: 1 to 151 of the patent application WO2017/036580, or fragments
or variants of these sequences. In this context, it is particularly
preferred that the 5'-UTR of the artificial RNA comprises or
consists of a corresponding RNA sequence of the nucleic acid
sequence according to SEQ ID NOs: 1 to 151 of the patent
application WO2017/036580.
[0140] The inventors observed that certain combinations of at least
one heterologous 5'-UTR and/or at least one heterologous 3'-UTR are
advantageously increasing the translation of the at least one
coding sequence operably linked to said 3'-UTR and/or 5'-UTR
encoding at least one antigenic peptide or protein derived from a
RSV F protein in the target tissue (e.g., muscle, dermis).
[0141] Accordingly it is preferred that the at least one
heterologous 5'-UTR as defined herein and the at least one
heterologous 3'-UTR as defined herein act synergistically to
increase production (that is translation) of antigenic peptide or
protein from the artificial RNA of the invention. These
advantageous combinations of 5'-UTR and 3-UTR are specified in the
following. Each of the abbreviation introduced below, namely "a-1",
"a-2", "a-3", "a-4", "a-5", "b-1", "b-2", "b-3", "b-4", "b-5",
"c-1", "c-2", "c-3" "c-4", "c-5", "d-1", "d-2", "d-3", "d-4",
"d-5", "e-1" "e-2" "e-3", "e-4", "e-5", "e-6", "f-1", "f-2", "f-3",
"f-4", "f-5, "g-1", "g-2", "g-3", "g-4", "g-5", "h-1", "h-2",
"h-3", "h-4", "h-5", i-1", "i-2", "i-3", are used throughout the
specification of the present invention and represent one
advantageous combination of 5'-UTR and/or 3'UTR of the
invention.
[0142] Accordingly, in preferred embodiments of the first aspect,
the artificial RNA of the invention comprises [0143] a-1. at least
one 5'-UTR derived from a 5'-UTR of a HSD174 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a PSMB3 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0144] a-2. at least one 5'-UTR derived from a 5'-UTR
of a NDUFA4 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a PSMB3 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0145] a-3. at least one
5'-UTR derived from a 5'-UTR of a SLC7A3 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a PSMB3 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0146] a-4. at least one 5'-UTR from a 5'-UTR of a
NOSIP gene, or from a corresponding RNA sequence, homolog, fragment
or variant thereof and at least one 3'-UTR derived from a 3'-UTR of
a PSMB3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof; or [0147] a-5. at least one 5'-UTR
derived from a 5'-UTR of a MP68 gene, or from a corresponding RNA
sequence, homolog, fragment or variant thereof and at least one
3'-UTR derived from a 3'-UTR of a PSMB3 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof;
or [0148] b-1. at least one 5'-UTR derived from a 5'-UTR of a
UBQLN2 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3-UTR derived from a
3'-UTR of a RPS9 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0149] b-2. at least one
5'-UTR derived from a 5'-UTR of a ASAH1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a RPS9 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0150] b-3. at least one 5'-UTR derived from a 5'-UTR
of a HSD17B4 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a RPS9 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0151] b-4. at least one
5'-UTR derived from a 5'-UTR of a HSD17B4 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a CASP1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0152] b-5. at least one 5'-UTR derived from a 5'-UTR
of a NOSIP gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a COX6B1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0153] c-1. at least one
5'-UTR derived from a 5'-UTR of a NDUFA4 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a RPS9 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0154] c-2. at least one 5'-UTR derived from a 5'-UTR
of a NOSIP gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a NDUFA1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0155] c-3. at least one
5'-UTR derived from a 5'-UTR of a NDUFA4 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a COX6B1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0156] c-4. at least one 5'-UTR derived from a 5'-UTR
of a NDUFA4 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a NDUFA1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0157] c-5. at least one
5'-UTR derived from a 5'-UTR of a ATP5A1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a PSMB3 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0158] d-1. at least one 5'-UTR derived from a 5'-UTR
of a RPL31 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a PSMB3 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0159] d-2. at least one
5'-UTR derived from a 5'-UTR of a ATP5A1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a CASP1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0160] d-3. at least one 5'-UTR derived from a 5'-UTR
of a SLC7A3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a GNAS gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0161] d-4. at least one
5'-UTR derived from a 5'-UTR of a HSD17B4 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a NDUFA1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0162] d-5. at least one 5'-UTR derived from a 5'-UTR
of a SLC7A3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a NDUFA1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0163] e-1. at least one
5'-UTR derived from a 5'-UTR of a TUBB4B gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a RPS9 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0164] e-2. at least one 5'-UTR derived from a 5'-UTR
of a RPL31 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a RPS9 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0165] e-3. at least one
5'-UTR derived from a 5'-UTR of a MP68 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a RPS9 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0166] e-4. at least one 5'-UTR derived from a 5'-UTR
of a NOSIP gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a RPS9 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0167] e-5. at least one
5'-UTR derived from a 5'-UTR of a ATP5A1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a RPS9 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0168] e-6. at least one 5'-UTR derived from a 5'-UTR
of a ATP5A1 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a COX6B1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0169] f-1. at least one
5'-UTR derived from a 5'-UTR of a ATP5A1 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a GNAS gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0170] f-2. at least one 5'-UTR derived from a 5'-UTR
of a ATP5A1 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a NDUFA1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0171] f-3. at least one
5'-UTR derived from a 5'-UTR of a HSD17B4 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a COX6B1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0172] f-4 at least one 5'-UTR derived from a 5'-UTR of
a HSD17B4 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3-UTR of a GNAS gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0173] f-5. at least one
5'-UTR derived from a 5'-UTR of a MP68 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a COX6B1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0174] g-1. at least one 5'-UTR derived from a 5'-UTR
of a MP68 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a NDUFA1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0175] g-2. at least one
5'-UTR derived from a 5'-UTR of a NDUFA4 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a CASP1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0176] g-3. at least one 5'-UTR derived from a 5'-UTR
of a NDUFA4 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a GNAS gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0177] g-4. at least one
5'-UTR derived from a 5'-UTR of a NOSIP gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a CASP1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0178] g-5. at least one 5'-UTR derived from a 5'-UTR
of a RPL31 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a CASP1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0179] h-1. at least one
5'-UTR derived from a 5'-UTR of a RPL31 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a COX6B1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0180] h-2. at least one 5'-UTR derived from a 5'-UTR
of a RPL31 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a GNAS gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0181] h-3. at least one
5'-UTR derived from a 5'-UTR of a RPL31 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a NDUFA1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0182] h-4. at least one 5'-UTR derived from a 5'-UTR
of a SLC7A3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3-UTR derived from a
3'-UTR of a CASP1 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof; or [0183] h-5. at least one
5'-UTR derived from a 5'-UTR of a SLC7A3 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a COX6B1 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof; or [0184] i-1. at least one 5'-UTR derived from a 5'-UTR
of a SLC7A3 gene, or from a corresponding RNA sequence, homolog,
fragment or variant thereof and at least one 3'-UTR derived from a
3'-UTR of a RPS9 gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof. [0185] i-2. at least one
5'-UTR derived from a 5'-UTR of a RPL32 gene, or from a
corresponding RNA sequence, homolog, fragment or variant thereof
and at least one 3'-UTR derived from a 3'-UTR of a ALB7 gene, or
from a corresponding RNA sequence, homolog, fragment or variant
thereof. [0186] i-3. at least one 3'-UTR derived from a 3'-UTR of a
alpha-globin gene gene, or from a corresponding RNA sequence,
homolog, fragment or variant thereof.
[0187] Suitably, the artificial RNA of the invention comprises at
least one coding sequence encoding at least one antigenic peptide
or protein derived from a RSV F protein as specified herein
operably linked to a 3'-UTR and a 5'-UTR selected from a-1
(HSD17B4/PSMB3), a-2 (Ndufa4/PSMB3), a-3 (SLC7A3/PSMB3), a-4
(NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RPS9), b-2
(ASAH1/RPS9), b-3 (HSD17B4/RPS9), b-4 (HSD17B4/CASP1), b-5
(NOSIP/COX6B1), c-1 (NDUFA4/RPS9), c-2 (NOSIP/NDUFA1), c-3
(NDUFA4/COX6B1), c-4 (NDUFA4/NDUFA1), c-5 (ATP5A1/PSMB3), d-1
(Rpl31/PSMB3), d-2 (ATP5A1/CASP1), d-3 (SLC7A3/GNAS), d-4
(HSD17B4/NDUFA1), d-5 (Sc7a3/Ndufa1), e-1 (TUBB4B/RPS9), e-2
(RPL31/RPS9), e-3 (MP68/RPS9), e-4 (NOSIP/RPS9), e-5 (ATP5A1/RPS9),
e-6 (ATP5A1/COX6B1), f-1 (ATP5A1/GNAS), f-2 (ATP5A1/NDUFA1), f-3
(HSD17B4/COX6B1), f-4 (HSD17B4/GNAS), f-5 (MP68/COX6B1), g-1
(MP68/NDUFA1), g-2 (NDUFA4/CASP1), g-3 (NDUFA4/GNAS), g-4
(NOSIP/CASP1), g-5 (RPL31/CASP1), h-1 (RPL31/COX6B1), h-2
(RPL31/GNAS), h-3 (RPL31/NDUFA1), h-4 (Slc7a3/CASP1), h-5
(SLC7A3/COX6B1), i-1 (SLC7A3/RPS9), i-2 (RPL32/ALB7), or i-3
(a-globin gene).
[0188] In particularly preferred embodiments of the first aspect,
the artificial RNA of the invention comprise UTR elements according
to a-1 (HSD17B4/PSMB3), a-4 (NDUFA4/PSMB3), c-1 (NDUFA4/RPS9), e-4
(NOSIP/RPS9), g-2 (NDUFA4/CASP1), i-2 (RPL32/ALB7), or i-3
(alpha-globin (muag)).
[0189] In a particularly preferred embodiment of the first aspect,
the artificial RNA of the invention comprises UTR elements
according to a-1 (HSD17B4/PSMB3).
[0190] The invention relates to an artificial RNA, preferably an
RNA suitable for vaccination, comprising at least one heterologous
5'-UTR as defined above and/or at least one heterologous 3'-UTR as
defined above and at least one coding sequence operably linked to
said 3'-UTR and/or 5'-UTR, wherein said coding sequence encodes at
least one antigenic peptide or protein derived from a Respiratory
syncytial virus ("RSV"), or a fragment or variant thereof.
[0191] As used herein, the term "Respiratory syncytial virus" or
the corresponding abbreviation "RSV" is not limited to a particular
virus strain, variant, serotype, or isolate, etc. comprising any
Respiratory syncytial virus of any origin.
[0192] According to various embodiments, the artificial RNA,
preferably the coding sequence of the artificial RNA comprises or
consists of a nucleic acid sequence that is derived from viruses,
with NCBI Taxonomy ID ("NCBI-ID") provided in List 1 below.
[0193] List 1: RSV Virus Strains:
[0194] Human orthopneumovirus, HRSV (NCBI-ID 11250); Human
respiratory syncytial virus A, HRSV-A, Respiratory syncytial virus
group A (NCBI-ID 208893); Human respiratory syncytial virus A
strain Long, Human respiratory syncytial virus (subgroup A/strain
Long) (NCBI-ID 11260); Human respiratory syncytial virus A2, Human
respiratory syncytial virus (strain A2), HRSVA (NCBI-ID 11259);
Human respiratory syncytial virus (strain RSB1734), (NCBI-ID
11253); Human respiratory syncytial virus (strain RSB5857) (NCBI-ID
11254); Human respiratory syncytial virus (strain RSB6190),
(NCBI-ID 11255); Human respiratory syncytial virus (strain
RSB6256), (NCBI-ID 11256); Human respiratory syncytial virus
(strain RSB642), (NCBI-ID 11252); Human respiratory syncytial virus
(strain RSB6614), (NCBI-ID 11257); Human respiratory syncytial
virus B, HRSV-B, Respiratory syncytial virus group B, (NCBI-ID
208895); Human Respiratory syncytial virus 9320 (NCBI-ID 253182);
Human respiratory syncytial virus B1 (NCBI-ID 79692); Human
respiratory syncytial virus (subgroup B/strain 18537), (NCBI-ID
11251); Human respiratory syncytial virus (subgroup B/strain 8/60),
(NCBI-ID 11258); Human respiratory syncytial virus S2, (NCBI-ID
410078); Human respiratory syncytial virus strain RSS-2, (NCBI-ID
11261); unclassified Human respiratory syncytial virus, (NCBI-ID
410233); Human respiratory syncytial virus (strain
RSP112/Sweden/02-03), (NCBI-ID 410237); Human respiratory syncytial
virus (strain RSP120/Sweden/02-03), (NCBI-ID 410238); Human
respiratory syncytial virus (strain RSP121/Sweden/02-03), (NCBI-ID
410239); Human respiratory syncytial virus (strain
RSP122/Sweden/02-03), (NCBI-ID 410247); Human respiratory syncytial
virus (strain RSP13/Sweden/02-03), (NCBI-ID 410241); Human
respiratory syncytial virus (strain RSP140/Sweden/02-03), (NCBI-ID
410248); Human respiratory syncytial virus (strain
RSP16/Sweden/02-03), (NCBI-ID 410242); Human respiratory syncytial
virus (strain RSP171/Sweden/02-03), (NCBI-ID 410246); Human
respiratory syncytial virus (strain RSP183/Sweden/02-03), (NCBI-ID
410249); Human respiratory syncytial virus (strain
RSP191/Sweden/02-03), (NCBI-ID 410240); Human respiratory syncytial
virus (strain RSP199/Sweden/02-03), (NCBI-ID 410250); Human
respiratory syncytial virus (strain RSP212/Sweden/02-03), (NCBI-ID
410251); Human respiratory syncytial virus (strain
RSP41/Sweden/02-03), (NCBI-ID 410234); Human respiratory syncytial
virus (strain RSP45/Sweden/02-03), (NCBI-ID 410235); Human
respiratory syncytial virus (strain RSP56/Sweden/02-03), (NCBI-ID
410243); Human respiratory syncytial virus (strain
RSP58/Sweden/02-03), (NCBI-ID 410236); Human respiratory syncytial
virus (strain RSP67/Sweden/02-03), (NCBI-ID 410244); Human
respiratory syncytial virus (strain RSP94/Sweden/02-03) (NCBI-ID
410245); Respiratory syncytial virus isolate RSV Memphis-37,
(strain Memphis-37) (NCBI-ID 12814).
[0195] In preferred embodiments of the invention, the at least one
antigenic peptide or protein is derived from a Respiratory
syncytial virus isolate RSV Memphis-37 (strain Memphis-37)
(NCBI-ID: 12814). Throughout the present invention, including the
information contained in the ST25 sequence listing, the
abbreviation "HRSV(Memphis-37)" is used for said particularly
preferred RSV virus.
[0196] In preferred embodiments of the invention, the at least one
antigenic peptide or protein is derived from a Human respiratory
syncytial virus A2, Human respiratory syncytial virus (strain A2)
(NCBI-ID: 11259). Throughout the present invention, including the
information contained in the ST25 sequence listing, the
abbreviation "HRSV(A2)" is used for said particularly preferred RSV
virus.
[0197] It has to be understood that the skilled person may also use
amino acid sequences and nucleic acid sequences derived from any
RSV strains provided in List 1 to adapt the teaching of the present
invention and to obtain RNA constructs, compositions, and vaccines
according to the invention.
[0198] In various embodiments, the at least one antigenic peptide
or protein may be selected from fusion protein (F), glycoprotein G,
short hydrophobic protein SH, matrix protein M, nucleoprotein N,
large polymerase L, M2-1 protein, M2-2 protein, phosphoprotein P,
non-structural protein NS1, or non-structural protein NS2 of
Respiratory syncytial virus (RSV) or a fragment, variant or
derivative thereof.
[0199] In particularly preferred embodiments of the first aspect,
the at least one antigenic peptide or protein is derived from an
RSV fusion (F) protein. In this context, the amino acid sequence of
the at least one antigenic peptide or protein may be selected from
any peptide or protein derived from RSV fusion protein F or from a
fragment, variant or derivative thereof.
[0200] RSV F protein is initially expressed (after infection of a
host cell) as a single polypeptide precursor, designated
full-length fusion protein F (herein referred to as "F0"). F0 forms
a trimer in the endoplasmic reticulum and is processed by a
cellular/host furin-like protease at two conserved sites,
generating, F1, F2, and Pep27 polypeptides. The Pep27 polypeptide
is excised and does not form part of the mature F protein. The F2
polypeptide originates from the N-terminal portion of the F0
precursor and links to the F1 polypeptide via two disulfide bonds.
The F1 polypeptide originates from the C-terminal portion of the F0
precursor and anchors the mature F protein in the membrane via a
transmembrane domain, which is linked to a cytoplasmic tail. Three
F2-F1 heterodimer units ("protomers") assemble to form a mature F
protein. Initially, the mature F protein is in a metastable form
(herein referred to as "pre-fusion conformation"). Upon triggering,
it undergoes a dramatic and irreversible conformational change
(herein referred to as "postfusion conformation") that fuses the
viral and target-cell membranes.
[0201] Accordingly, the artificial RNA of the first aspect,
preferably the artificial RNA suitable for vaccination, encodes at
least one antigenic peptide or protein derived from a RSV F protein
or a fragment or variant thereof.
[0202] In preferred embodiments, at least one antigenic peptide or
protein derived from a RSV F protein may be derived from any one of
the following amino acid sequences (NCBI Protein Accession numbers)
provided in List 2 below.
[0203] List 2: NCBI Protein Accession Numbers of RSV Fusion (F)
Proteins:
[0204] Accession No. Protein, AJF44801.1, AJF44759.1, AJF44661.1,
AJF44602.1, 2207424A, AAB38520.1, AAB38517.1, AAB38519.1,
AAB38518.1, AVQ93587.1, AVQ93599.1, AVQ93571.1, AVQ93568.1,
AVQ93589.1, AVQ93597.1, AVQ93563.1, AVQ93594.1, AVQ93606.1,
AVQ93601.1, AVQ93562.1, AVQ93561.1, AVQ93607.1, AVQ93588.1,
AVQ93575.1, AVQ93468.1, AVQ93467.1, AVQ93590.1, AVQ93552.1,
AVQ93556.1, AVQ93471.1, AVQ93458.1, AVQ93494.1, AVQ93470.1,
AVQ93489.1, AVQ93542.1, AVQ93472.1, AVQ93514.1, AVQ93485.1,
AVQ93533.1, AVQ93481.1, AVQ93546.1, AVQ93512.1, AVQ93554.1,
AVQ93551.1, AVQ93547.1, AVQ93558.1, AVQ93461.1, AVQ93500.1,
AVQ93426.1, AVQ93398.1, AVQ93401.1, AVQ93361.1, AVQ93408.1,
AVQ93443.1, AVQ93429.1, AVQ93359.1, AVQ93365.1, AVQ93366.1,
AVQ93402.1, AVQ93377.1, AVQ93412.1, AVQ93391.1, AVQ93457.1,
AVQ93372.1, AVQ93455.1, AVQ93364.1, AVQ93378.1, AVQ93393.1,
AVQ93362.1, AVQ93585.1, ART28504.1, AVQ93404.1, AOS49123.1,
AOS48496.1, AMT78271.1, AHX57174.1, AHW81390.1, AHV81506.1,
AFX60128.1, AFX60129.1, AEQ63389.1, ARB66328.1, ANZ80034.1,
AMN91253.1, P03420.1, A1008046.1, NP_056863.1, AFX60234.1,
AFX60231.1, AFX60232.1, AFX60222.1, AFX60219.1, AFX60215.1,
AFX60214.1, AFX60212.1, AFX60208.1, AFX60202.1, AFX60220.1,
AFX60213.1, AFX60190.1, AFX60187.1, AFX60201.1, AFX60173.1,
AFX60169.1, AFX60162.1, AFX60156.1, AFX60151.1, AFX60150.1,
AFX60148.1, AFX60141.1, AFX60127.1, AFX60137.1, AFX60135.1,
AFV46420.1, AFX60200.1, AFV46419.1, AFV46417.1, AFV46413.1,
AFV46414.1, AFV46410.1, AFV46403.1, AFV46409.1, AFP99061.1,
AFM95400.1, AFV46401.1, AFP99064.1, AFM95376.1, AFX60138.1,
AFP99060.1, AFM95365.1, AFM55563.1, AFM55530.1, AFM55442.1,
AFM55420.1, AFM55552.1, AFM55365.1, AFP99059.1, AFM95385.1,
AFM55354.1, AFM55343.1, AFM55387.1, AFM55299.1, AFM55288.1,
AFM55266.1, AFM55277.1, AFM55255.1, AFM55222.1, AFM55211.1,
AFI25262.1, AFD34266.1, AFM55332.1, AFD34264.1, AFD34262.1,
AFD34265.1, AFD34261.1, AFD34260.1, AFD34259.1, AEQ63641.1,
AEQ63487.1, AEQ63520.1, AEQ63378.1, AEQ63367.1, 4CCF_A, AEQ63334.1,
AEO45949.1, AEO45939.1, AEQ63312.1, AEQ63586.1, AEO45919.1,
AEO45909.1, AEO45889.1, AEO45879.1, AEO45869.1, AEO45929.1,
AEO45850.1, AEO45859.1, AEQ63444.1, AEO23054.1, AEO23052.1,
AEO23051.1, AEC32087.1, ADZ95785.1, AEC32085.1, ADZ95784.1,
ADZ95783.1, ADZ95782.1, ADZ95781.1, ADZ95779.1, ADZ95780.1,
ADZ95777.1, ADZ95778.1, ADZ95776.1, ADZ95775.1, ACY68435.1,
ACO83302.1, AB35685.1, AFI25251.1, AAX23994.1, AAQ97026.1,
AAR14266.1, AAQ97027.1, AAQ97030.1, AAQ97028.1, AAC57027.1,
AAQ97029.1, AAQ97031.1, AAM68160.1, AAM44851.1, P11209.2, P13843.1,
AAO72325.1, AAM68157.1, CAA26143.1, 1512372A, AAB86664.1,
AAO72324.1, AAB82446.1, AAO72323.1, AAM68154.1, AAA47410.1,
P12568.1, ARB07894.1, AGG39517.1, BBC54612.1, BBC54636.1,
BBC54627.1, BBC54621.1, BBC54570.1, BBC54579.1, BBC54595.1,
BBC54555.1, BBC54552.1, BBC54564.1, BBC54581.1, BBC54571.1,
BBC54553.1, BBC54565.1, BBC54245.1, BBC54243.1, BBC54238.1,
BBC54242.1, BBC54239.1, BBC54235.1, BBC54234.1, BBC54236.1,
BBC54244.1, BBC54186.1, BBC54178.1, BBC54202.1, BBC54151.1,
BBC54142.1, BBC54134.1, BBC54170.1, BBC54210.1, BBC54169.1,
BBC54213.1, BBC54203.1, BBC54160.1, BBC54163.1, BBC54215.1,
BBC54156.1, BBC54179.1, BBC54150.1, BBC54207.1, BBC54194.1,
BBC54149.1, BBC54138.1, BBC54199.1, BBC54220.1, BBC54181.1,
BBC54132.1, BBC54146.1, BBC54122.1, BBC54124.1, BBB35202.1,
BBB35201.1, BBB35192.1, BBB35193.1, BBB35199.1, BBB35184.1,
BBB35126.1, BBB35133.1, BBB35130.1, BBB35160.1, BBB35162.1,
BBB35181.1, BBB35165.1, BBB35121.1, BBB35138.1, BBB35176.1,
BBB35142.1, BBB35136.1, BBB35153.1, BBB35115.1, BBB35150.1,
BBB35097.1, BBB35109.1, BBB35094.1, BBB35183.1, BBB35104.1,
BBB35099.1, BBB35188.1, AKA45871.1, ASV65838.1, AGG39373.1,
AII22107.1, AGG39400.1, AGG39457.1, ARR29240.1, ARR29251.1,
ARR29189.1, ARR29207.1, AUH15164.1, ATV81343.1, AUC68654.1,
AUC68577.1, AUC68566.1, AUC68555.1, AUC68478.1, AUC68445.1,
AUC68500.1, AUC68522.1, AUC68291.1, AUC68149.1, AUC68094.1,
AMA67097.1, AMA66866.1, AMA66580.1, AIZ95750.1, AIZ95541.1,
AIZ95519.1, AHY21419.1, AHY21331.1, AHY21165.1, AHY21143.1,
AHY21132.1, AGG39478.1, ATV93506.1, ATV93509.1, AIZ95717.1,
ATV81354.1, ART28317.1, BBA57890.1, BBA57901.1, ASZ70099.1,
ART28361.1, AQX36844.1, ASK05520.1, ART28427.1, ART28339.1,
ART28297.1, ART28328.1, ARQ15966.1, ARN61507.1, ARA15413.1,
AGG39394.1, APW78845.1, APW78867.1, APW78900.1, APW78878.1,
APW78779.1, APW78702.1, APW78713.1, APW78680.1, APW78658.1,
APW78647.1, APW78636.1, AOS48870.1, APW78614.1, AMT78905.1,
AMT77402.1, AOZ15479.1, AGN28484.1, AHX57240.1, AHX57031.1,
AOS48980.1, AOS48848.1, AOS48815.1, AOS48738.1, AOS49068.1,
AOS48727.1, AOS48716.1, AOS48683.1, AOS48551.1, AOS48485.1,
AOS48518.1, AOS48441.1, AOS48397.1, AOS48375.1, AOS48353.1,
AOS48287.1, AOS48320.1, AOS48265.1, AOS48254.1, AOS48221.1,
ANZ79638.1, ALC74025.1, AHV81286.1, AOD40888.1, AOD40516.1,
AOD40214.1, AOD40125.1, AOD40104.1, AOD40082.1, AOD39908.1,
AJF44826.1, AJF44506.1, AJF44535.1, ANZ80463.1, ANZ80408.1,
ANZ80397.1, ANZ80386.1, ANZ80331.1, ANZ80320.1, ANZ80364.1,
ANZ80276.1, ANZ80221.1, ANZ80188.1, ANZ80144.1, ANZ80133.1,
ANZ80111.1, ANZ80122.1, ANZ80056.1, ANZ80012.1, ANZ80067.1,
ANZ79979.1, ANZ79935.1, ANZ79990.1, ANZ79902.1, ANZ79759.1,
ANZ79715.1, ANZ79671.1, AMT79718.1, AMT79586.1, AMT79553.1,
AMT79542.1, AMT79476.1, AMT79388.1, AMT79201.1, AMT79190.1,
AMT79157.1, AMT79124.1, AMT79091.1, AMT79047.1, AMT79014.1,
AMT79003.1, AMT78872.1, AMT78832.1, AMT78689.1, AMT78645.1,
AMT78513.1, AMT78480.1, AMT78447.1, AMT78392.1, AMT78194.1,
AMT78084.1, AMT78051.1, AMT77963.1, AMT77908.1, AMT77776.1,
AMT77710.1, AMT77424.1, AMN91385.1, AMN91264.1, AMN91242.1,
AHX57504.1, CUS01881.1, CUS01880.1, CUS01877.1, CUS01874.1,
CUS01870.1, CUS01875.1, AMA67350.1, AMA67251.1, AMA67262.1,
AMA67229.1, AMA67196.1, AMA67130.1, AMA67185.1, AMA67163.1,
AMA67086.1, AMA67075.1, AMA66998.1, AMA66987.1, AMA66976.1,
AMA66965.1, AMA66921.1, AMA66844.1, AMA66833.1, AMA66624.1,
AMA66613.1, AMA66591.1, AMA66569.1, AMA66547.1, AMA66503.1,
AMA66492.1, AMA66481.1, AMA66448.1, AMA66415.1, AMA66393.1,
AMA66360.1, AGG39553.1, AGG39469.1, AJZ70144.1, AJZ70166.1,
AJZ70155.1, AJZ70133.1, AJZ70067.1, AJZ70001.1, AJZ69990.1,
AJZ69968.1, AJZ69946.1, AJZ69913.1, AJZ69880.1, AJZ69847.1,
AJZ69869.1, AJZ69770.1, AJZ69748.1, AJZ69726.1, AJZ69682.1,
AJZ69671.1, AJZ69704.1, AJZ69660.1, AJZ69715.1, AJZ69627.1,
AJZ69616.1, AJZ69638.1, AJO16077.1, AJO16055.1, AKE31881.1,
AKE31882.1, AKE31878.1, AJF44835.1, AJF44790.1, AJF44737.1,
AJF44716.1, AJF44725.1, AJF44643.1, AJF44628.1, AJF44624.1,
AJF44613.1, AJF44566.1, AJF44555.1, AJF44526.1, AJF44517.1,
AKA45882.1, AKA45849.1, AHA83913.1, AGN28715.1, AHA83902.1,
AHA83837.1, AHA83826.1, AHA83705.1, AHA83630.1, AGG39505.1,
AIY70242.1, AIY70198.1, AII30203.1, AHY21463.1, AHY21397.1,
AHY21320.1, AHY21298.1, AHY21287.1, AHY21276.1, AHY21254.1,
AHY21199.1, AHY21176.1, AHX57537.1, AHX57471.1, AHX57427.1,
AHX57042.1, AIZ95981.1, AIZ95893.1, AIZ95871.1, AIZ95816.1,
AIZ95772.1, AIZ95629.1, AIZ95673.1, AIZ95596.1, AIZ95585.1,
AIZ95552.1, AEQ63553.2, AEQ63542.2, AEQ63575.1, AEQ63531.1,
AEQ63498.1, AEQ63422.1, AEQ63411.1, AHX57570.1, AHX57152.1,
AHX57064.1, AHX57009.1, AHX56987.1, AHV82100.1, AHV82001.1,
AHV81891.1, AHV81880.1, AHV81836.1, AHV81682.1, AHV81649.1,
AHV81484.1, AHV81462.1, AHV81385.1, AHV81363.1, AHV81330.1,
AHV81253.1, AHV81154.1, AHV81122.1, AHV81089.1, AHV81012.1,
AHV80957.1, AHV80880.1, AHV80869.1, AHV80836.1, AHV80803.1,
AGG39559.1, AGG39562.1, AGG39556.1, AGG39550.1, AGG39547.1,
AGG39544.1, AGG39541.1, AGG39529.1, AGG39526.1, AGG39523.1,
AGG39514.1, AGG39502.1, AGG39499.1, AGG39496.1, AGG39493.1,
AGG39484.1, AGG39490.1, AGG39487.1, AGG39475.1, AGG39472.1,
AGG39466.1, AGG39463.1, AGG39454.1, AGG39442.1, AGG39439.1,
AGG39436.1, AGG39415.1, AGG39403.1, AGG39397.1, AGG39391.1,
AGG39379.1, AHL84194.1, AHA84012.1, AHJ60043.1, BAO49770.1,
BAO49766.1, BAO49767.1, AHA84034.1, AHA84023.1, AHA83990.1,
AHA83957.1, AHA83924.1, AHA83891.1, AHA83880.1, AHA83782.1,
AHA83760.1, AHA83694.1, AGT75357.1, AGN92848.1, AGN28792.1,
AGN28781.1, AGN28759.1, AGN28748.1, AGN28693.1, AGN28638.1,
AGN28627.1, AGN28539.1, AGN28528.1, AGN28462.1, AGN28440.1,
AGL96787.1, AGL96786.1, AGL96784.1, AAS93662.1, AAS93657.1,
AAS93656.1, AAS93659.1, AAS93660.1, AAS93663.1, AAS93664.1,
AAS93655.1, CUS01869.1, AHG54517.1, ASF87348.1, ASF87341.1,
ASF87344.1, ASF87351.1, ASF87338.1, ASF87342.1, ASF87352.1,
ASF87337.1, ASF87336.1, AEQ98756.1, AEQ98757.1, AEQ98755.1,
AEQ98752.1, AEQ98753.1, AEQ98747.1, ASF87325.1, ASF87326.1, 5W23_A,
5EA3_F, 5UDD_A, 5EA8_F, AHG54458.1, AEN74947.1, AHG54485.1,
AHG54477.1, AHG54451.1, AHG54463.1, AHG54445.1, AEO12131.1,
AEN74945.1, AEN74944.1, ASF87335.1, AHG54515.1, AHA61605.1,
AHV81660.1, AHG54441.1 or AIY60640.1.
[0205] In the context of the present invention, "RSV F protein",
"RSV fusion protein (F)", "RSV F", or "F" may be understood in its
broadest sense and refers to F0 (F polypeptide precursor), F1, F2
and Pep27 polypeptides, F2-F1 heterodimer, or the mature F protein
(comprising three F2-F1 heterodimers), or fragments and variants of
any of these. Accordingly, the term "peptide or protein derived
from a RSV fusion (F) protein" refers to a peptide, protein,
fragment or variant derived from e.g. F0 (F protein polypeptide
precursor), F1, F2 and Pep27 polypeptides, F2-F1 heterodimer, or
the mature F protein. Additionally, the term "peptide or protein
derived from a RSV fusion (F) protein" refers to peptide, protein,
fragment or variant derived from "RSV F protein" or "RSV fusion
protein (F)" as defined above which may be genetically engineered
to e.g. to lack certain protein elements (e.g. the cytoplasmic
tail, the furin cleavage site, Pep27) or e.g. comprise additional
elements (e.g., linker elements, heterologous signal peptides
etc.). For example, the term "peptide or protein derived from a RSV
fusion (F) protein" refers to peptide, protein, fragment or variant
derived from F0, F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2,
F_DSCav1_mut3, F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6,
F_DSCav1_mut7, F_DSCav1_mut8, F_DSCav1_mut0, F-del_DSCav1,
F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3,
F-del_DSCav1_mut4, F-del_DSCav1_mut5, F-del_DSCav1_mut6,
F-del_DSCav1_mut7, F-del_DSCav1_mut8, F-del_DSCav1_mut0 (for
explanation of the constructs see Table 1). Particularly suitable F
protein variants that may be encoded by the RNA of the first aspect
are specified in the following and are provided in Table 1.
[0206] It has to be noted that where reference is made to amino
acid (aa) residues and their position in an RSV F protein, any
numbering used herein--unless stated otherwise--relates to the
position of the respective aa residue in a corresponding F0
precursor protein of HRSV(A2) (SEQ ID NO: 68) or a corresponding F0
precursor protein of HRSV(Memphis-37) (SEQ ID NO: 8937 or 11726)
wherein position "1" corresponds to the first aa residue, i.e. the
aa residue at the N-terminus of a HRSV(A2) F0 precursor protein or
a HRSV(Memphis-37) F0 precursor protein.
[0207] In preferred embodiments, the at least one coding sequence
of the RNA of the first aspect encodes at least one antigenic
peptide or protein from RSV F protein, wherein RSV F protein is a
full-length F protein (F0) or an F protein with deleted C-terminus
(F-del), or a fragment or a variant thereof.
[0208] In this context, any RSV F full-length protein (precursor
protein, referred to as "F0") may be used as suitable antigen and
may preferably be derived from any NCBI Protein Accession numbers
provided in List 2 or may be chosen from any one of SEQ ID NOs: 68,
8279-8967 or 11726. In preferred embodiments of the invention, the
full-length F protein (F0) of HRSV(A2) (SEQ ID NO: 68) is suitably
used, see overview Table 1. In other preferred embodiments of the
invention, the full-length F protein (F0) of HRSV(Memphis-37) (SEQ
ID NO: 8937 or 11726) is suitably used, see overview Table 1.
[0209] In this context, any RSV F with deleted C-terminus (F-del)
may be used as suitable antigen and may preferably be derived from
any NCBI Protein Accession numbers provided in List 2 or may be
chosen from any one of SEQ ID NOs: 483, 8968-9683, or 12095. An
example of such a deletion mutant is the RSV-Fdel554-574 protein
according to (Oomens et al. 2006. J. Virol. 80(21):10465-77) where
aa residue 554-574 of the full-length F0 protein are removed.
Deletion of the main part of the cytoplasmic tail (aa 554-574) of
F0 leads to improved intracellular trafficking/cell surface
transport in vitro and increases cell surface expression of RSV F
significantly. Increased cell surface presentation results in
improved B cell recognition (in line with published data; see
WO2015024668). In preferred embodiments of the invention, F protein
with deleted C-terminus, herein referred to as "F-del" (SEQ ID NO:
483, 9653 or 12095) is suitably used, see overview Table 1. In the
light of high level of structural conservation of the RSV F protein
among different RSV strains (see e.g. Hause et al., 2017, PLOS ONE
12(6): e0180623), the deletion of aa 554-574 is applicable to
different RSV F protein sequences of different RSV isolates.
[0210] In particularly preferred embodiments, the artificial RNA of
the first aspect encodes least one antigenic peptide or protein
derived from RSV F protein, wherein said RSV F protein is designed
to stabilize the antigen in pre-fusion conformation. A pre-fusion
conformation is particularly advantageous in the context of an
efficient RSV vaccine, as several potential epitopes for
neutralizing antibodies are merely accessible in said protein
conformation.
[0211] In several embodiments, the RSV F protein includes one or
more amino acid substitutions that stabilize the F protein in the
pre-fusion conformation, for example, substitutions that stabilize
the membrane distal portion of the F protein (including the
N-terminal region of the F1 polypeptide) in the pre-fusion
conformation. For example, the amino acid substitution can
introduce a non-natural disulfide bond or can be a cavity-filling
amino acid substitution.
[0212] Accordingly, in several embodiments, a preferred RSV F
protein includes S155C and S290C substitutions that form a
non-natural disulfide bond that stabilizes the protein in a
pre-fusion conformation; that is, in a conformation that
specifically binds to one or more pre-fusion specification
antibodies, and/or presents a suitable antigenic site that is
present on the pre-fusion conformation but not in the postfusion
conformation of RSV F protein. In further embodiments, the
recombinant RSV F protein can additionally include F, L, W, Y, H,
or M substitution at position 190, position 207, or positions 190
and 207.
[0213] An example of an RSV F protein designed to stabilize the
antigen in pre-fusion conformation is RSV F protein comprising a
DSCav1 mutation (S155C, S290C, S190F, and V207L), or a fragment or
a variant thereof. Such RSV F DSCav1 proteins have been described
in the art (W2014160463).
[0214] Accordingly, in particularly preferred embodiments, the
artificial RNA of the first aspect encodes least one antigenic
peptide or protein derived from RSV F protein, wherein the RSV F
protein comprises a DSCav1 mutation (S155C, S290C, S190F, and
V207L), or a fragment or a variant thereof.
[0215] It has to be understood in the context of the invention that
any RSV F may be mutated at positions S155C, S290C, S190F, and
V207L to stabilize the protein in the pre-fusion conformation and
may be suitably used in the context of the invention. Accordingly,
any NCBI Protein Accession numbers provided above, or any protein
selected from SEQ ID NOs: 68, 8279-8967, 483, 8968-9683, 12095, or
11726 or fragments or variants thereof can be chosen by the skilled
person to introduce such amino acid changes according to S155C,
S290C, S190F, and V207L to generate various RSV F DSCav1
proteins.
[0216] In preferred embodiments, RSV F full-length protein
(precursor protein, "F0") of HRSV(A2) (SEQ ID NO: 68) is used to
introduce S155C, S290C, S190F, and V207L amino acid changes,
leading to an amino acid sequence according to SEQ ID NO: 898. Such
a RSV F protein is herein referred to as "F0_DSCav1" throughout the
present invention (see overview Table 1 (preferred RSV F protein
antigen designs)).
[0217] In other preferred embodiments, RSV F_del protein (deleted
cytoplasmic tail (aa 554-574)) of HRSV(A2) (SEQ ID NO: 483) is used
to introduce S155C, S290C, S190F, and V207L amino acid changes,
leading to an amino acid sequence according to SEQ ID NO: 1267.
Such a RSV F protein is herein referred to as "F-del_DSCav1"
throughout the present invention (see overview Table 1 (preferred
RSV F protein antigen designs)).
[0218] In preferred embodiments, RSV F full-length protein
(precursor protein, "F0") of HRSV(Memphis-37) (SEQ ID NO: 8937 or
11726) is used to introduce S155C, S290C, S190F, and V207L amino
acid changes, leading to an amino acid sequence according to SEQ ID
NO: 12464. Such a RSV F protein is herein referred to as
"FO_DSCav1" throughout the present invention (see overview Table 1
(preferred RSV F protein antigen designs)).
[0219] In other preferred embodiments, RSV F_del protein (deleted
cytoplasmic tail (aa 554-574)) of HRSV(Memphis-37) (SEQ ID NO: 9653
or 12095) is used to introduce S155C, S290C, S190F, and V207L amino
acid changes, leading to an amino acid sequence according to SEQ ID
NO: 12833. Such a RSV F protein is herein referred to as
"F-del_DSCav1" throughout the present invention (see overview Table
1 (preferred RSV F protein antigen designs).
[0220] In preferred embodiments, the at least one antigenic peptide
or protein may be an engineered protein comprising the two
subunits, F1 and F2 of mature F as a single polypeptide chain,
wherein F2 and F1 are preferably connected via a linker (GS).
Examples of said engineered F2-linker-F1 fusion proteins (e.g.,
F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553)) have been
described in the art (Joyce, M. Gordon, et al. "Iterative
structure-based improvement of a fusion-glycoprotein vaccine
against RSV." Nature structural & molecular biology 23.9
(2016): 811; WO2017172890). Said F2-linker-F1 RSV F proteins lack
aa104-144 (comprising the furine cleavage site and Pep27) and
comprise a linker element between F2 polypeptide and F1 polypeptide
(e.g. GSlinker). Said F2-linker-F1 proteins may show superior
properties in terms of stability and/or antigenicity.
[0221] Accordingly, in preferred embodiments, the RSV F protein
comprises the two subunits F2 and F1 fused into a single
polypeptide chain, wherein F2 and F1 are connected via a linker
element, preferably a GS linker as specified herein, preferably
generating a stable F2-linker-F1 proteins.
[0222] Preferably, said F2-linker-F1 fusion proteins, e.g.
F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) additionally
comprise a DScav1 mutation as outlined above (herein referred to as
"mut0"; e.g., SEQ ID NOs: 3850, 4219 or 13940, 14309).
[0223] In particularly preferred embodiments, the RSV F protein may
additionally comprise at least one further mutation selected from
(S46G, A149C, S215P, Y458C, K465Q), (S46G, E92D, A149C, S215P,
Y458C, K465Q), (S46G, N671, E92D, A149C, S215P, Y458C, K465Q),
(A149C, Y458C), (N183GC, N428C), (Q98C, Q361C, S46G, E92D, L95M,
S215P, I217P, I221M, R429K, K465Q), (Q98C, Q361C, L95M, I221M,
R429K), or (N183GC, N428C, S46G, N671, E92D, S215P, K465Q) or a
fragment or a variant thereof.
[0224] In particularly preferred embodiments, said F2-linker-F1
proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553)) may
additionally comprise, preferably in addition to the DSCav1
mutation, at least one mutation selected from S46G, A149C, S215P,
Y458C, K465Q, herein referred to as "mut1", e.g., SEQ ID NOs: 1636,
2005, or 14678, 15047; or S46G, E92D, A149C, S215P, Y458C, K465Q,
herein referred to as "mut2", e.g., SEQ ID NOs: 2374, 2743 or
15416, 15785; or S46G, N671, E92D, A149C, S215P, Y458C, K465Q,
herein referred to as "mut3", e.g., SEQ ID NOs: 3112, 3481 or
13202, 13571, or a fragment or a variant of any of these (see
overview Table 1 (preferred RSV F protein antigen designs)).
[0225] In other embodiments, said F2-linker-F1 proteins
(F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553)) may additionally
comprise, preferably in addition to the DSCav1 mutation, at least
one mutation selected from A149C, Y458C, herein referred to as
"mut4", e.g., SEQ ID NOs: 4588, 4957 or 16154, 16523; or N183GC,
N428C, herein referred to as "mut5", e.g., SEQ ID NOs: 5326, 5695
or 16892, 17261; or Q98C, Q361C, S46G, E92D, L95M, S215P, I217P,
I221M, R429K, K465Q, herein referred to as "mut6", e.g., SEQ ID
NOs: 6064, 6433 or 17630, 17999; or Q98C, Q361C, L95M, I221M,
R429K, herein referred to as "mut7", e.g., SEQ ID NOs: 6802, 7171
or 18368, 18737; or N183GC, N428C, S46G, N671, E92D, S215P, K465Q,
herein referred to as "mut8", e.g., SEQ ID NOs: 7540, 7909 or
19106, 19475, or a fragment or a variant thereof.
[0226] It has to be understood in the context of the invention that
any RSV F may be adapted in a way that the two subunits, F1 and F2
are comprised in a single polypeptide chain, wherein F2 and F1 may
be connected via a linker, e.g. a (GS) linker to enhance stability
of the protein as explained for mutations "mut1", "mut2" and
"mut3", e.g. by deleting aa104-144 of the F0 polypeptide chain (as
explained above), by introducing a linker element between F2 and F1
as explained above, and by introducing suitable amino acid
substitutions as explained above. Accordingly, any NCBI Protein
Accession numbers provided above (see List 2), or any protein
selected from SEQ ID NOs: 68, 8279-8967, 483, 8968-9683, 11726,
12095 or fragments or variants thereof can be adapted by the
skilled person to generate F2-linker-F1 fusion proteins as outlined
herein, and may additionally be adapted by introducing (S46G,
A149C, S215P, Y458C, K465Q), (S46G, E92D, A149C, S215P, Y458C,
K465Q), (S46G, N671, E92D, A149C, S215P, Y458C, K465Q), (A149C,
Y458C), (N183GC, N428C), (Q98C, Q361C, S46G, E92D, L95M, S215P,
I217P, I221M, R429K, K465Q), (Q98C, Q361C, L95M, I221M, R429K), or
(N183GC, N428C, S46G, N671, E92D, S215P, K465Q) aa substitutions
and/or a DSCav1 mutation. Moreover, apart from using a GS linker as
outlined above, the skilled person may of course choose between
various known linker elements to arrive at similar likewise
suitable RSV F protein variants (e.g. selected from SEQ ID NOs:
117-162 of the patent application WO2017/172890 or fragments or
variants of these sequences, or selected from SEQ ID NOs: 1509-1565
of the patent application WO2017/081082, or fragments or variants
of these sequences).
[0227] In preferred embodiments, F2-linker-F1 proteins
(F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a
DSCav-1 mutation as outlined above, herein referred to as
F_DSCav1_mut0 or F-del_DSCav1_mut0. In preferred embodiments,
F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or
F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined
above, and additionally an amino acid substitution mut1 as defined
above, herein referred to as F_DSCav1_mut1 or F-del_DSCav1_mut1. In
preferred embodiments, F2-linker-F1 proteins
(F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a
DSCav-1 mutation as outlined above, and additionally an amino acid
substitution mut2 as defined above, herein referred to as
F_DSCav1_mut2 or F-del_DSCav1_mut2. In preferred embodiments,
F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or
F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined
above, and additionally an amino acid substitution mut3 as defined
above, herein referred to as F_DSCav1_mut3 or F-del_DSCav1_mut3. In
preferred embodiments, F2-linker-F1 proteins
(F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a
DSCav-1 mutation as outlined above, and additionally an amino acid
substitution mut4 as defined above, herein referred to as
F_DSCav1_mut4 or F-del_DSCav1_mut4. In preferred embodiments,
F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or
F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined
above, and additionally an amino acid substitution mut5 as defined
above, herein referred to as F_DSCav1_mut5 or F-del_DSCav1_mut5. In
preferred embodiments, F2-linker-F1 proteins
(F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a
DSCav-1 mutation as outlined above, and additionally an amino acid
substitution mut6 as defined above, herein referred to as
F_DSCav1_mut6 or F-del_DSCav1_mut6 . In preferred embodiments,
F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or
F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined
above, and additionally an amino acid substitution mut7 as defined
above, herein referred to as F_DSCav1_mut7 or F-del_DSCav1_mut7. In
preferred embodiments, F2-linker-F1 proteins
(F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a
DSCav-1 mutation as outlined above, and additionally an amino acid
substitution mut8 as defined above, herein referred to as F_DSCav1
mut8 or F-del_DSCav1 mut8.
[0228] A detailed description of particularly preferred RSV F
proteins is provided in Table 1.
[0229] In Table 1 all references made to amino acid (aa) residues
and their position in an RSV F protein relates to the position of
the respective aa residue in a corresponding F0 precursor protein
of HRSV(A2) (SEQ ID NO: 68) or HRSV(Memphis-37) (SEQ ID NO: 8937 or
11726). Moreover, the abbreviations for suitable RSV F protein
antigen designs in Table 1 are used throughout the description of
the invention (e.g., "F0", "F-del", "F0_DSCav1", "F-del_DSCav1",
"F_DSCav1_mut1", "F-del_DSCav1_mut1", "F_DSCav1_mut2",
"F-del_DSCav1_mut2", "F_DSCav1_mut3", "F-del_DSCav1_mut3",
"F_DSCav1_mut4", "F-del_DSCav1_mut4", "F_DSCav1_mut5",
"F-del_DSCav1_mut5", "F_DSCav1_mut6", "F-del_DSCav1_mut6",
"F_DSCav1_mut7", "F-del_DSCav1_mut7 ", "F_DSCav1_mut8",
"F-del_DSCav1_mut8", "F_DSCav1_mut0", "F-del_DSCav1_mut0"). Column
A of Table 1 provides protein SEQ ID NOs of respective RSV F
protein antigen designs derived from HRSV(A2); Column B of Table 1
provides protein SEQ ID NOs of respective RSV F protein antigen
designs derived from HRSV(Memphis-37). Notably, the description of
the invention explicitly includes the information provided under
<223> identifier of the ST25 sequence listing of the present
application.
TABLE-US-00001 TABLE 1 Preferred RSV F protein antigen designs
Antigen Name Protein design description A B F0 aa1-574, 68 8937,
full-length RSV F0 precursor 11726 F-del aa1-553, 483 9653,
deletion of aa 554-574 of the C-terminus 12095 F0_DSCav1 aa1-574,
898 12464 aa substitutions: S155C, S290C, S190F, and V207L
F-del_DSCav1 aa1-553, 1267 12833 deletion of aa 554-574 of the
C-terminus, aa substitutions: S155C, S290C, S190F, and V207L
F_DSCav1_mut0 aa1-103 - GS(linker) - aa145-574 3850 13940
F2-linker-F1 construct aa substitutions: S155C, S290C, S190F, and
V207L F-del_DSCav1_mut0 aa1-103 - GS(linker) - aa145-553 4219 14309
deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct aa
substitutions: S155C, S290C, S190F, and V207L F_DSCav1_mut1 aa1-103
- GS(linker) - aa145-574, 1636 14678 F2-linker-F1 construct, aa
substitutions: S155C, S290C, S190F, and V207L; S46G, A149C, S215P,
Y458C, K465Q F-del_DSCav1_mut1 aa1-103 - GS(linker) - aa145-553
2005 15047 deletion of aa 554-574 of the C-terminus, F2-linker-F1
construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G,
A149C, S215P, Y458C, K465Q F_DSCav1_mut2 aa1-103 - GS(linker) -
aa145-574 2374 15416 F2-linker-F1 construct, aa substitutions:
S155C, S290C, S190F, and V207L; S46G, E92D, A149C, S215P, Y458C,
K465Q F-del_DSCav1_mut2 aa1-103 - GS(linker) - aa145-553 2743 15785
deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct,
aa substitutions: S155C, S290C, S190F, and V207L; S46G, E92D,
A149C, S215P, Y458C, K465Q F_DSCav1_mut3 aa1-103 - GS(linker) -
aa145-574 3112 13202 F2-linker-F1 construct, aa substitutions:
S155C, S290C, S190F, and V207L; S46G, N67I, E92D, A149C, S215P,
Y458C, K465Q F-del_DSCav1_mut3 aa1-103 - GS(linker) - aa145-553
3481 13571 deletion of aa 554-574 of the C-terminus, F2-linker-F1
construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G,
N67I, E92D, A149C, S215P, Y458C, K465Q F_DSCav1_mut4 aa1-103 -
GS(linker) - aa145-574 4588 16154 F2-linker-F1 construct, aa
substitutions: S155C, S290C, S190F, and V207L; A149C, Y458C
F-del_DSCav1_mut4 aa1-103 - GS(linker) - aa145-553 4957 16523
deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct,
aa substitutions: S155C, S290C, S190F, and V207L; A149C, Y458C
F_DSCav1_mut5 aa1-103 - GS(linker) - aa145-574 5326 16892
F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and
V207L; N183GC, N428C F-del_DSCav1_mut5 aa1-103 - GS(linker) -
aa145-553 5695 17261 deletion of aa 554-574 of the C-terminus,
F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and
V207L; N183GC, N428C F_DSCav1_mut6 aa1-103 - GS(linker) - aa145-574
6064 17630 F2-linker-F1 construct, aa substitutions: S155C, S290C,
S190F, and V207L; Q98C, Q361C, S46G, E92D, L95M, S215P, I217P,
I221M, R429K, K465Q F-del_DSCav1_mut6 aa1-103 - GS(linker) -
aa145-553 6433 17999 deletion of aa 554-574 of the C-terminus,
F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and
V207L; Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K,
K465Q F_DSCav1_mut7 aa1-103 - GS(linker) - aa145-574 6802 18368
F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and
V207L; Q98C, Q361C, L95M, I221M, R429K F-del_DSCav1_mut7 aa1-103 -
GS(linker) - aa145-553 7171 18737 deletion of aa 554-574 of the
C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C,
S190F, and V207L; Q98C, Q361C, L95M, I221M, R429K F_DSCav1_mut8
aa1-103 - GS(linker) - aa145-574 7540 19106 F2-linker-F1 construct,
aa substitutions: S155C, S290C, S190F, and V207L; N183GC, N428C,
S46G, N67I, E92D, S215P, K465Q F-del_DSCav1_mut8 aa1-103 -
GS(linker) - aa145-553 7909 19475 deletion of aa 554-574 of the
C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C,
S190F, and V207L; N183GC, N428C, S46G, N67I, E92D, S215P, K465Q
[0230] In particularly preferred embodiments, the artificial RNA
according to the first aspect encodes at least one antigenic
peptide or protein derived from a RSV fusion (F) protein, wherein
the RSV F protein is selected from F0, F-del, F0_DSCav1,
F_DSCav1_mut0, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3,
F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6, F_DSCav1_mut7,
F_DSCav1_mut8, F-del_DSCav1, F-del_DSCav1_mut0, F-del_DSCav1_mut1,
F-del_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut4,
F-del_DSCav1_mut5, F-del_DSCav1_mut6, F-del_DSCav1_mut7,
F-del_DSCav1_mut8, or a fragment or a variant thereof (see e.g.
Table 1).
[0231] Particularly preferred and advantageous in the context of
the invention are RSV F proteins selected from F-del_DSCav1,
F-del_DSCav1_mut0, F-del_DSCav1_mut1, F-del_DSCav1_mut2,
F-del_DSCav1_mut3, F-del_DSCav1_mut4, F-del_DSCav1_mut5,
F-del_DSCav1_mut6, F-del_DSCav1_mut7, F-del_DSCav1_mut8 or a
fragment or a variant thereof (see e.g. Table 1).
[0232] In preferred embodiments, the artificial RNA of the first
aspect comprises at least one coding sequence encoding at least one
antigenic peptide or protein comprising or consisting of at least
one amino acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
19475 (see e.g. Table 1), or 8279-9683 or a fragment or variant of
any of these sequences. Additional information regarding each of
these suitable amino acid sequences encoding RSV proteins may also
be derived from the or 99% identical to any one of SEQ ID NO: 68,
483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219,
4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726,
12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785,
13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368,
18737, 19106, sequence listing, in particular from the details
provided therein under identifier <223> as explained in the
following.
[0233] In preferred embodiments, the artificial RNA of the first
aspect comprises at least one coding sequence encoding at least one
antigenic peptide or protein comprising or consisting of at least
one amino acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636,
2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695,
6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464, 12833,
13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154,
16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 (see
e.g. Table 1) or a fragment or variant of any of these sequences.
Additional information regarding each of these suitable amino acid
sequences encoding RSV proteins may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223> as explained in the following.
[0234] In particularly preferred embodiments, the artificial RNA of
the first aspect comprises at least one coding sequence encoding at
least one antigenic peptide or protein comprising or consisting of
at least one amino acid sequences being identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to any one of SEQ ID NO: 1267, 2005,
2743, 3481, 4219, 4957, 5695, 6433, 7171, 7909, 12833, 14309,
15047, 15785, 13571, 16523, 17261, 17999, 18737, 19475 (see e.g.
Table 1) or a fragment or variant of any of these sequences.
Additional information regarding each of these suitable amino acid
sequences encoding RSV proteins may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223> as explained in the following.
[0235] In other embodiments, the artificial RNA according to the
first aspect comprises at least one coding sequence encoding at
least one of the amino acid sequences being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 8279-9683
a fragment or variant of any of these sequences. Additional
information regarding each of these suitable amino acid sequences
encoding RSV proteins may also be derived from the sequence
listing, in particular from the details provided therein under
identifier <223> as explained in the following.
[0236] In other embodiments, the artificial RNA as defined herein
comprises at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV comprising or
consisting of at least one amino acid sequences being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:
1-1428 of patent application WO2014/160463 or a fragment or variant
of any of these sequences. In this context, SEQ ID NOs: 1-1428 of
patent application WO2014/160463 and the disclosure related thereto
are herewith incorporated by reference.
[0237] In other embodiments, the artificial RNA as defined herein
comprises at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV comprising or
consisting of at least one amino acid sequences being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:
1-11 of patent application WO2015/024668 or a fragment or variant
of any of these sequences. In this context, SEQ ID NOs: 1-11 of
patent application WO2015/024668 and the disclosure related thereto
are herewith incorporated by reference.
[0238] In other embodiments, the artificial RNA as defined herein
comprises at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV comprising or
consisting of at least one amino acid sequences being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO:
3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 243, or 245 of
patent application WO2017/070622 or a fragment or variant of any of
these sequences. In this context SEQ ID NO: 3, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 243, or 245, of patent application
WO2017/070622 and the disclosure related thereto are herewith
incorporated by reference.
[0239] In other embodiments, the artificial RNA as defined herein
comprises at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV comprising or
consisting of at least one amino acid sequences being identical or
at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:
1-65, 81-95, 110-116 of patent application WO2017/172890 or a
fragment or variant of any of these sequences. In this context SEQ
ID NOs: 1-65, 81-95, 110-116, of patent application WO2017/172890
and the disclosure related thereto are herewith incorporated by
reference.
[0240] According to another preferred embodiment, the artificial
RNA of the invention encodes at least one antigenic peptide or
protein as defined above and may additionally encode at least one
further heterologous peptide or protein element.
[0241] Suitably, the at least one further peptide or protein
element may promote secretion of the encoded antigenic peptide or
protein of the invention (e.g. via secretory signal sequences),
promote anchoring of the encoded antigenic peptide or protein of
the invention in the plasma membrane (e.g. via transmembrane
elements), promote formation of antigen complexes (e.g. via
multimerization domains), promote virus-like particle formation
(VLP forming sequence). In addition, the artificial nucleic acid
sequence according to the present invention may additionally encode
peptide linker elements, self-cleaving peptides, immunologic
adjuvant sequences or dendritic cell targeting sequences. Suitable
multimerization domains may be selected from the list of amino acid
sequences according to SEQ ID NOs: 1116-1167 of the patent
application WO2017/081082, or fragments or variants of these
sequences. Trimerization and tetramerization elements may be
selected from e.g. engineered leucine zippers (engineered
.alpha.-helical coiled coil peptide that adopt a parallel trimeric
state), fibritin foldon domain from enterobacteria phage T4,
GCN4pII, GCN4-pLI, and p53. In that context, fibritin foldon domain
from enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53 are
preferred. Suitable transmembrane elements may be selected from the
list of amino acid sequences according to SEQ ID NOs: 1228-1343 of
the patent application WO2017/081082, or fragments or variants of
these sequences. Suitable VLP forming sequences may be selected
from the list of amino acid sequences according to SEQ ID NOs:
1168-1227 of the patent application WO2017/081082, or fragments or
variants of these sequences. Suitable peptide linkers may be
selected from the list of amino acid sequences according to SEQ ID
NOs: 1509-1565 of the patent application WO2017/081082, or
fragments or variants of these sequences. Suitable self-cleaving
peptides may be selected from the list of amino acid sequences
according to SEQ ID NOs: 1434-1508 of the patent application
WO2017/081082, or fragments or variants of these sequences.
Suitable immunologic adjuvant sequences may be selected from the
list of amino acid sequences according to SEQ ID NOs: 1360-1421 of
the patent application WO2017/081082, or fragments or variants of
these sequences. Suitable dendritic cell (DCs) targeting sequences
may be selected from the list of amino acid sequences according to
SEQ ID NOs: 1344-1359 of the patent application WO2017/081082, or
fragments or variants of these sequences. Suitable secretory signal
peptides may be selected from the list of amino acid sequences
according to SEQ ID NOs: 1-1115 and SEQ ID NO: 1728 of the patent
application WO2017/081082, or fragments or variants of these
sequences. On nucleic acid level, any nucleic acid sequence (e.g.
RNA sequence) may be selected which encodes such amino acid
sequences. In this context, the disclosure of WO2017/081082 is
herewith incorporated by reference. The heterologous secretory
signal sequence may increase the secretion of the encoded antigenic
peptide or protein.
[0242] According to embodiments, the secretory signal sequence
comprises an amino acid sequence being identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to any one of SEQ ID NOs: 21329-21362 or
a fragment or variant of any of these sequences. Additional
information regarding each of these suitable amino acid sequences
encoding secretory signal sequences may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223>.
[0243] According to preferred embodiments, the artificial nucleic
acid, particularly the artificial RNA comprises at least one coding
sequence encoding at least one antigenic peptide or protein derived
from RSV as defined herein, preferably derived from RSV F protein,
or fragments and variants thereof. In that context, any coding
sequence encoding at least one antigenic peptide or protein derived
from RSV, preferably derived from RSV F protein, or fragments and
variants thereof may be understood as suitable coding sequence and
may therefore be comprised in the artificial RNA of the first
aspect.
[0244] In preferred embodiments, the artificial RNA of the first
aspect may comprise or consist of at least one coding sequence
encoding at least one antigenic peptide or protein derived from RSV
F protein as defined herein, preferably encoding any one of SEQ ID
NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850,
4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909,
11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416,
15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999,
18368, 18737, 19106, 19475 or fragments of variants thereof. It has
to be understood that, on nucleic acid level, any nucleic acid
sequence, in particular, any RNA sequence which encodes an amino
acid sequences being identical to SEQ ID NO: 68, 483, 898, 1267,
1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326,
5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464,
12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571,
16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106,
19475 or fragments or variants thereof, or any nucleic acid
sequence (e.g. DNA sequence, RNA sequence) which encodes amino acid
sequences being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one
of SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112,
3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171,
7540, 7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047,
15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630,
17999, 18368, 18737, 19106, 19475 or fragments or variants thereof,
may be selected and may accordingly be understood as suitable
coding sequence and may therefore be comprised in the artificial
RNA of the first aspect of the invention.
[0245] In other embodiments, the artificial RNA of the first aspect
may comprise or consist of at least one coding sequence encoding
any one of SEQ ID NOs: 8279-9683 or fragments of variants thereof.
It has to be understood that, on nucleic acid level, any nucleic
acid sequence, in particular, any RNA sequence which encodes an
amino acid sequences being identical to SEQ ID NOs: 8279-9683 or
fragments or variants thereof, or any nucleic acid sequence (e.g.
DNA sequence, RNA sequence) which encodes amino acid sequences
being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID
NOs: 8279-9683 or fragments or variants thereof, may be selected
and may accordingly be understood as suitable coding sequence and
may therefore be comprised in the artificial RNA of the first
aspect of the invention.
[0246] In other embodiments, the artificial RNA of the first aspect
may comprise or consist of at least one coding sequence encoding
any one of SEQ ID NOs: 1-1428 of patent application WO2014/160463
or fragments of variants thereof. It has to be understood that, on
nucleic acid level, any nucleic acid sequence, in particular, any
RNA sequence which encodes an amino acid sequences being identical
to SEQ ID NOs: 1-1428 of patent application WO2014/160463 or
fragments or variants thereof, or any nucleic acid sequence (e.g.
DNA sequence, RNA sequence) which encodes amino acid sequences
being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID
NOs: 1-1428 of patent application WO2014/160463 or fragments or
variants thereof, may be selected and may accordingly be understood
as suitable coding sequence and may therefore be comprised in the
artificial RNA of the first aspect of the invention.
[0247] In other embodiments, the artificial RNA of the first aspect
may comprise or consist of at least one coding sequence encoding
any one of SEQ ID NOs: 1-11 of patent application WO2015/024668 or
fragments of variants thereof. It has to be understood that, on
nucleic acid level, any nucleic acid sequence, in particular, any
RNA sequence which encodes an amino acid sequences being identical
to SEQ ID NOs: 1-11 of patent application WO2015/024668 or
fragments or variants thereof, or any nucleic acid sequence (e.g.
DNA sequence, RNA sequence) which encodes amino acid sequences
being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID
NOs: 1-11 of patent application WO2015/024668 or fragments or
variants thereof, may be selected and may accordingly be understood
as suitable coding sequence and may therefore be comprised in the
artificial RNA of the first aspect of the invention.
[0248] In other embodiments, the artificial RNA of the first aspect
may comprise or consist of at least one coding sequence encoding
any one of SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 243, or 245 of patent application WO2017/070622 or
fragments of variants thereof. It has to be understood that, on
nucleic acid level, any nucleic acid sequence, in particular, any
RNA sequence which encodes an amino acid sequences being identical
to SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
243, or 245 of patent application WO2017/070622 or fragments or
variants thereof, or any nucleic acid sequence (e.g. DNA sequence,
RNA sequence) which encodes amino acid sequences being at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 243, or 245 of patent
application WO2017/070622 or fragments or variants thereof, may be
selected and may accordingly be understood as suitable coding
sequence and may therefore be comprised in the artificial RNA of
the first aspect of the invention.
[0249] In other embodiments, the artificial RNA of the first aspect
may comprise or consist of at least one coding sequence encoding
any one of SEQ ID NOs: 1-65, 81-95, 110-116 of patent application
WO2017/172890 or fragments of variants thereof. It has to be
understood that, on nucleic acid level, any nucleic acid sequence,
in particular, any RNA sequence which encodes an amino acid
sequences being identical to SEQ ID NOs: 1-65, 81-95, 110-116 of
patent application WO2017/172890 or fragments or variants thereof,
or any nucleic acid sequence (e.g. DNA sequence, RNA sequence)
which encodes amino acid sequences being at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to any one of SEQ ID NOs: 1-65, 81-95, 110-116 of
patent application WO2017/172890 or fragments or variants thereof,
may be selected and may accordingly be understood as suitable
coding sequence and may therefore be comprised in the artificial
RNA of the first aspect of the invention.
[0250] Suitably, in particularly preferred embodiments, the
artificial RNA of the first aspect comprises a coding sequence
located between said 5'-UTR and said 3'-UTR, preferably downstream
of said 5'-UTR and upstream of said 3'-UTR.
[0251] In preferred embodiments, the artificial RNA of the first
aspect comprises a coding sequence that comprises at least one of
the nucleic acid sequences being identical or at least 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to SEQ ID NOs: 69-482, 484-897, 899-1266,
1268-1635, 1637-2004, 2006-2373, 2375-2742, 2744-3111, 3113-3480,
3482-3849, 3851-4218, 4220-4587, 4589-4956, 4958-5325, 5327-5694,
5696-6063, 6065-6432, 6434-6801, 6803-7170, 7172-7539, 7541-7908,
7910-8277, 8278, 11727-12094, 12096-12463, 12465-12832,
12834-13201, 13941-14308, 14310-14677, 14679-15046, 15048-15415,
15417-15784, 15786-16153, 13203-13570, 13572-13939, 16155-16522,
16524-16891, 16893-17260, 17262-17629, 17631-17998, 18000-17998,
18369-18736, 18738-19105, 19107-19474, 19476-19843, 21363-21706 or
a fragment or a fragment or variant of any of these sequences (see
also Table 3-6). Additional information regarding each of these
suitable nucleic acid sequences encoding may also be derived from
the sequence listing, in particular from the details provided
therein under identifier <223>.
[0252] In particularly preferred embodiments, the artificial RNA of
the first aspect comprises a coding sequence that comprises at
least one of the nucleic acid sequences being identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to SEQ ID NOs: 69-77, 484-492,
899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751,
3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965,
5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179,
7541-7548, 7910-7917, 21363-21384, 11727-11734, 12096-12103,
12465-12472, 12834-12841, 13941-13948, 14310-14317, 14679-14686,
15048-15055, 15417-15424, 15786-15793, 13203-13210, 13572-13579,
16155-16162, 16524-16531, 16893-16900, 17262-17269, 17631-17638,
18000-18007, 18369-18376, 18738-18745, 19107-19114, 19476-19483,
21389-21410 or a fragment or a fragment or variant of any of these
sequences (see also Table 3 and 4). Additional information
regarding each of these suitable nucleic acid sequences encoding
may also be derived from the sequence listing, in particular from
the details provided therein under identifier <223>.
[0253] In other embodiments, the artificial RNA of the first aspect
comprises a coding sequence that comprises at least one of the
nucleic acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NOs: 383-388 of patent application
WO2014/160463 or a fragment or a fragment or variant of any of
these sequences.
[0254] In other embodiments, the artificial RNA of the first aspect
comprises a coding sequence that comprises at least one of the
nucleic acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NOs: 12-22 of patent application
WO2015/024668 or a fragment or a fragment or variant of any of
these sequences.
[0255] In other embodiments, the artificial RNA of the first aspect
comprises a coding sequence that comprises at least one of the
nucleic acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NOs: 1, 2, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 242, 244, 246, 257, 258-280 of patent application
WO2017/070622 or a fragment or a fragment or variant of any of
these sequences.
[0256] In other embodiments, the artificial RNA of the first aspect
comprises a coding sequence that comprises at least one of the
nucleic acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to SEQ ID NOs: 96-99 of patent application
WO2017/172890 or a fragment or a fragment or variant of any of
these sequences.
[0257] According to preferred embodiments, the artificial RNA is a
modified and/or stabilized artificial RNA.
[0258] According to preferred embodiments, the artificial RNA of
the present invention may thus be provided as a "stabilized
artificial RNA" that is to say an RNA showing improved resistance
to in vivo degradation and/or an artificial RNA showing improved
stability in vivo, and/or an artificial RNA showing improved
translatability in vivo. In the following, specific suitable
modifications in this context are described which are suitably to
"stabilize" the artificial RNA.
[0259] Such stabilization may be effected by providing a "dried
RNA" and/or a "purified RNA" as specified herein. Alternatively or
in addition to that, such stabilization can be effected, for
example, by a modified phosphate backbone of the artificial RNA of
the present invention. A backbone modification in connection with
the present invention is a modification in which phosphates of the
backbone of the nucleotides contained in the RNA are chemically
modified. Nucleotides that may be preferably used in this
connection contain e.g. a phosphorothioate-modified phosphate
backbone, preferably at least one of the phosphate oxygens
contained in the phosphate backbone being replaced by a sulfur
atom. Stabilized RNAs may further include, for example: non-ionic
phosphate analogues, such as, for example, alkyl and aryl
phosphonates, in which the charged phosphonate oxygen is replaced
by an alkyl or aryl group, or phosphodiesters and
alkylphosphotriesters, in which the charged oxygen residue is
present in alkylated form. Such backbone modifications typically
include, without implying any limitation, modifications from the
group consisting of methylphosphonates, phosphoramidates and
phosphorothioates (e.g. cytidine-5-O-(1-thiophosphate)).
[0260] In the following, suitable modifications are described that
are capable of "stabilizing" the artificial RNA of the
invention.
[0261] According to embodiments, the artificial RNA according to
the invention is a modified artificial RNA, wherein the
modification refers to chemical modifications comprising backbone
modifications as well as sugar modifications or base
modifications.
[0262] In this context, a modified artificial RNA as defined herein
may contain nucleotide analogues/modifications, e.g. backbone
modifications, sugar modifications or base modifications. A
backbone modification in connection with the present invention is a
modification, in which phosphates of the backbone of the
nucleotides contained in a nucleic acid, e.g. an artificial RNA,
are chemically modified. A sugar modification in connection with
the present invention is a chemical modification of the sugar of
the nucleotides of the RNA as defined herein. Furthermore, a base
modification in connection with the present invention is a chemical
modification of the base moiety of the nucleotides of the RNA. In
this context, nucleotide analogues or modifications are preferably
selected from nucleotide analogues which are applicable for
transcription and/or translation.
[0263] In particularly preferred embodiments of the present
invention, the nucleotide analogues/modifications which may be
incorporated into a modified nucleic acid or particularly into a
modified RNA as described herein are preferably selected from
2-amino-6-chloropurineriboside-5'-triphosphate,
2-Aminopurine-riboside-5'-triphosphate;
2-aminoadenosine-5'-triphosphate,
2'-Amino-2'-deoxycytidine-triphosphate,
2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate,
2'-Fluorothymidine-5'-triphosphate,
2'-O-Methyl-inosine-5'-triphosphate 4-thiouridine-5'-triphosphate,
5-aminoallylcytidine-5'-triphosphate,
5-aminoallyluridine-5'-triphosphate,
5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate,
5-Bromo-2'-deoxycytidine-5'-triphosphate,
5-Bromo-2'-deoxyuridine-5'-triphosphate,
5-iodocytidine-5'-triphosphate,
5-Iodo-2'-deoxycytidine-5'-triphosphate,
5-iodouridine-5'-triphosphate,
5-Iodo-2'-deoxyuridine-5'-triphosphate,
5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate,
5-Propynyl-2'-deoxycytidine-5'-triphosphate,
5-Propynyl-2'-deoxyuridine-5'-triphosphate,
6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate,
6-chloropurineriboside-5'-triphosphate,
7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate,
8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate,
benzimidazole-riboside-5'-triphosphate,
N1-methyladenosine-5'-triphosphate,
N1-methylguanosine-5'-triphosphate,
N6-methyladenosine-5'-triphosphate,
06-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate,
or puromycin-5'-triphosphate, xanthosine-5'-triphosphate.
Particular preference is given to nucleotides for base
modifications selected from the group of base-modified nucleotides
consisting of 5-methylcytidine-5'-triphosphate,
7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate,
and pseudouridine-5'-triphosphate, pyridin-4-one ribonucleoside,
5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,
4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,
3-methyluridine, 5-carboxymethyl-uridine,
1-carboxymethyl-pseudouridine, 5-propynyl-uridine,
1-propynyl-pseudouridine, 5-taurinomethyluridine,
1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,
1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,
1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,
2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-dihydropseudouridine, 2-methoxyuridine,
2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and
4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine,
N4-methylcytidine, 5-hydroxymethylcytidine,
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine,
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, and
4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,
6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,
7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,
7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,
1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,
N6-(cis-hydroxyisopentenyl)adenosine,
2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,
N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,
2-methylthio-N6-threonyl carbamoyladenosine,
N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and
2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine,
7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,
6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,
N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and
N2,N2-dimethyl-6-thio-guanosine, 5'-O-(1-thiophosphate)-adenosine,
5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine,
5'-O-(1-thiophosphate)-uridine,
5'-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine,
2-thio-cytidine, alpha-thio-cytidine, Pseudo-iso-cytidine,
5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine,
5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine,
6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine,
5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha-thio-guanosine,
6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine,
7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine,
N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine,
N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine,
7-deaza-adenosine. Particularly preferred and suitable in the
context of the invention are pseudouridine (.psi.),
N1-methylpseudouridine (m1.psi.), 5-methylcytosine, and
5-methoxyuridine. Accordingly, the artificial RNA as defined herein
may comprise at least one modified nucleotide selected from
pseudouridine (.psi.), N1-methylpseudouridine (m1.psi.),
5-methylcytosine, and 5-methoxyuridine.
[0264] In preferred embodiments, the artificial RNA of the
invention comprises at least one coding sequence, wherein the at
least one coding sequence is a pseudouridine (.psi.) modified
coding sequence.
[0265] Accordingly, in preferred embodiments, the artificial RNA of
the invention, or the at least one coding sequence, comprises a
nucleic acid sequence wherein at least one or more than one,
preferably wherein all uracil nucleotides are replaced by
pseudouridine (.psi.) nucleotides
[0266] In further preferred embodiments, the artificial RNA of the
invention comprises at least one coding sequence, wherein the at
least one coding sequence is a N1-methylpseudouridine (m1.psi.)
modified coding sequence.
[0267] Accordingly, in preferred embodiments, the artificial RNA of
the invention, or the at least one coding sequence, comprises a
nucleic acid sequence wherein at least one or more than one,
preferably wherein all uracil nucleotides are replaced by
N1-methylpseudouridine (m1.psi.) nucleotides
[0268] In preferred embodiments, the artificial RNA of the
invention comprises at least one coding sequence, wherein the at
least one coding sequence is a codon modified coding sequence.
[0269] In preferred embodiments, the at least one coding sequence
of the invention is a codon modified coding sequence, wherein the
amino acid sequence encoded by the at least one codon modified
coding sequence is preferably not being modified compared to the
amino acid sequence encoded by the corresponding wild type coding
sequence.
[0270] The term "codon modified coding sequence" relates to coding
sequences that differ in at least one codon (triplets of
nucleotides coding for one amino acid) compared to the
corresponding wild type coding sequence. Suitably, a codon modified
coding sequence in the context of the invention may show improved
resistance to in vivo degradation and/or improved stability in
vivo, and/or improved translatability in vivo. Codon modifications
in the broadest sense make use of the degeneracy of the genetic
code wherein multiple codons may encode the same amino acid and may
be used interchangeably (cf. Table 2) to optimize/modify the coding
sequence for in vivo applications as outlined above.
[0271] In particularly preferred embodiments of the first aspect,
the at least one sequence is a codon modified coding sequence,
wherein the codon modified coding sequence is selected from C
maximized coding sequence, CAI maximized coding sequence, human
codon usage adapted coding sequence, G/C content modified coding
sequence, and G/C optimized coding sequence, or any combination
thereof, or any combination thereof.
[0272] According to preferred embodiments, the artificial RNA of
the invention may be modified, wherein the C content of the at
least one coding sequence may be increased, preferably maximized,
compared to the C content of the corresponding wild type coding
sequence (herein referred to as "C maximized coding sequence"). The
amino acid sequence encoded by the C maximized coding sequence of
the RNA is preferably not modified as compared to the amino acid
sequence encoded by the respective wild type nucleic acid coding
sequence. The generation of a C maximized nucleic acid sequences
may suitably be carried out using a modification method according
to WO2015/062738. In this context, the disclosure of WO2015/062738
is included herewith by reference. Throughout the disclosure of the
invention, including the <223> identifier of the sequence
listing, C maximized coding sequences of suitable RSV nucleic acid
sequences are indicated by the abbreviation "opt2".
[0273] According to embodiments, the artificial RNA of the present
invention may be modified, wherein the G/C content of the at least
one coding sequence of the invention may be modified compared to
the G/C content of the corresponding wild type coding sequence
(herein referred to as "G/C content modified coding sequence"). In
this context, the terms "G/C optimization" or "G/C content
modification" relate to a nucleic acid, preferably an artificial
nucleic acid of the invention that comprises a modified, preferably
an increased number of guanosine and/or cytosine nucleotides as
compared to the corresponding wild type nucleic acid sequence. Such
an increased number may be generated by substitution of codons
containing adenosine or thymidine nucleotides by codons containing
guanosine or cytosine nucleotides. If the enriched G/C content
occurs in a coding sequence of DNA or RNA, it makes use of the
degeneracy of the genetic code. In particular, in case of RNA,
sequences having an increased G (guanosine)/C (cytosine) content
are more stable than sequences having an increased A (adenosine)/U
(uracil) content. The amino acid sequence encoded by the G/C
content modified coding sequence of the nucleic acid sequence is
preferably not modified as compared to the amino acid sequence
encoded by the respective wild type nucleic acid coding sequence.
Preferably, the G/C content of the coding sequence of the
artificial nucleic acid sequence, e.g. the RNA sequence of the
present invention is increased by at least 10%, preferably by at
least 20%, more preferably by at least 30%, most preferably by at
least 40% compared to the G/C content of the coding sequence of the
corresponding wild type nucleic acid sequence (e.g. RNA sequence),
which codes for a RSV antigen as defined herein or a fragment or
variant thereof.
[0274] According to preferred embodiments, the artificial RNA of
the present invention may be modified, wherein the G/C content of
the at least one coding sequence of the invention may be optimized
compared to the G/C content of the corresponding wild type coding
sequence (herein referred to as "G/C content optimized coding
sequence"). "Optimized" in that context refers to a coding sequence
wherein the G/C content is preferably increased to the essentially
highest possible G/C content. The amino acid sequence encoded by
the G/C content optimized coding sequence of the nucleic acid
sequence is preferably not modified as compared to the amino acid
sequence encoded by the respective wild type nucleic acid coding
sequence. The generation of a G/C content optimized nucleic RNA
sequence may suitably be carried out using a G/C content
optimization method according to WO2002/098443. In this context,
the disclosure of WO2002/098443 is included in its full scope in
the present invention. Throughout the disclosure of the invention,
including the <223> identifier of the sequence listing, G/C
optimized coding sequences of suitable RSV nucleic acid sequences
are indicated by the abbreviation "opt1, opt5, opt6, opt11".
[0275] According to embodiments, the artificial RNA of the
invention may be modified, wherein the codons in the at least one
coding sequence of the invention may be adapted to human codon
usage (herein referred to as "human codon usage adapted coding
sequence"). Codons encoding the same amino acid occur at different
frequencies in a subject, e.g. a human. Accordingly, the coding
sequence of the artificial RNA is preferably modified such that the
frequency of the codons encoding the same amino acid corresponds to
the naturally occurring frequency of that codon according to the
human codon usage e.g. as shown in Table 2. For example, in the
case of the amino acid Ala, the wild type coding sequence is
preferably adapted in a way that the codon "GCC" is used with a
frequency of 0.40, the codon "GCT" is used with a frequency of
0.28, the codon "GCA" is used with a frequency of 0.22 and the
codon "GCG" is used with a frequency of 0.10 etc. (see Table 2).
Accordingly, such a procedure (as exemplified for Ala) is applied
for each amino acid encoded by the coding sequence of the
artificial nucleic acid of the invention to obtain sequences
adapted to human codon usage. Throughout the disclosure of the
invention, including the <223> identifier of the sequence
listing, human codon usage adapted coding sequences of suitable RSV
nucleic acid sequences are indicated by the abbreviation
"opt3".
TABLE-US-00002 TABLE 2 Human codon usage table with frequencies
indicated for each amino acid Amino acid codon frequency Ala GCG
0.10 Ala GCA 0.22 Ala GCT 0.28 Ala GCC* 0.40 Cys TGT 0.42 Cys TGC*
0.58 Asp GAT 0.44 Asp GAC* 0.56 Glu GAG* 0.59 Glu GAA 0.41 Phe TTT
0.43 Phe TTC* 0.57 Gly GGG 0.23 Gly GGA 0.26 Gly GGT 0.18 Gly GGC*
0.33 His CAT 0.41 His CAC* 0.59 Ile ATA 0.14 Ile ATT 0.35 Ile ATC*
0.52 Lys AAG* 0.60 Lys AAA 0.40 Leu TTG 0.12 Leu TTA 0.06 Leu CTG*
0.43 Leu CTA 0.07 Leu CTT 0.12 Leu CTC 0.20 Met ATG* 1 Asn AAT 0.44
Asn AAC* 0.56 Pro CCG 0.11 Pro CCA 0.27 Pro CCT 0.29 Pro CCC* 0.33
Gln CAG* 0.73 Gln CAA 0.27 Arg AGG 0.22 Arg AGA* 0.21 Arg CGG 0.19
Arg CGA 0.10 Arg CGT 0.09 Arg CGC 0.19 Ser AGT 0.14 Ser AGC* 0.25
Ser TCG 0.06 Ser TCA 0.15 Ser TCT 0.18 Ser TCC 0.23 Thr ACG 0.12
Thr ACA 0.27 Thr ACT 0.23 Thr ACC* 0.38 Val GTG* 0.48 Val GTA 0.10
Val GTT 0.17 Val GTC 0.25 Trp TGG* 1 Tyr TAT 0.42 Tyr TAC* 0.58
Stop TGA* 0.61 Stop TAG 0.17 Stop TAA 0.22 *most frequent human
codon
[0276] According to embodiments, the artificial RNA of the present
invention may be modified, wherein the codon adaptation index (CAI)
may be increased or preferably maximised in the at least one coding
sequence of the invention (herein referred to as "CAI maximized
coding sequence"). Accordingly, it is preferred that all codons of
the wild type nucleic acid sequence that are relatively rare in
e.g. a human cell are exchanged fora respective codon that is
frequent in the e.g. a human cell, wherein the frequent codon
encodes the same amino acid as the relatively rare codon. Suitably,
the most frequent codons are used for each encoded amino acid (see
Table 2, most frequent human codons are marked with asterisks).
Suitably, the artificial RNA of the invention comprises at least
one coding sequence, wherein the codon adaptation index (CAI) of
the at least one coding sequence is at least 0.5, at least 0.8, at
least 0.9 or at least 0.95. Most preferably, the codon adaptation
index (CAI) of the at least one coding sequence is 1. For example,
in the case of the amino acid Ala, the wild type coding sequence is
adapted in a way that the most frequent human codon "GCC" is always
used for said amino acid. Accordingly, such a procedure (as
exemplified for Ala) is applied for each amino acid encoded by the
coding sequence of the artificial RNA of the invention to obtain
CAI maximized coding sequences. Throughout the disclosure of the
invention including the <223> identifier of the sequence
listing, CAI maximized coding sequences of suitable RSV nucleic
acid sequences are indicated by the abbreviation "opt4".
[0277] Accordingly, in a particularly preferred embodiment, the
artificial RNA of the first aspect comprises at least one coding
sequence comprising a codon modified nucleic acid sequence which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codon
modified nucleic acid sequence selected from the group consisting
of SEQ ID NOs: 70-77, 485-492, 899-906, 1268-1275, 1637-1644,
2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858,
4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072,
6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 11728-11734,
12097-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317,
14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210,
13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269,
17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114,
19476-19483, 21363-21384, 21389-21410 or a fragment or variant of
any of these sequences (see also Table 3 and 4). Additional
information regarding each of these suitable nucleic acid sequences
encoding may also be derived from the sequence listing, in
particular from the details provided therein under identifier
<223>.
[0278] In particularly preferred embodiment, the artificial RNA of
the first aspect comprises at least one coding sequence comprising
a codon modified nucleic acid sequence which is identical or at
least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the
G/C optimized or G/C content modified nucleic acid sequence
according to the SEQ ID NOs: 70-71, 75-77, 485-486, 490-492,
899-900, 904-906, 1268-1269, 1273-1275, 1637-1638, 1642-1644,
2006-2007, 2011-2013, 2375-2376, 2380-2382, 2744-2745, 2749-2751,
3113-3114, 3118-3120, 3482-3483, 3487-3489, 3852, 4221, 4590, 4959,
5328, 5697, 6066, 6435, 6804, 7173, 7542, 7911, 3856-3858,
4225-4227, 4594-4596, 4963-4965, 5332-5334, 5701-5703, 6070-6072,
6439-6441, 6808-6810, 7177-7179, 7546-7548, 7915-7917, 11728,
11732-11734, 12097, 12101-12103, 12465, 12466, 12470-12472, 12834,
12835, 12839-12841, 13941, 13942, 13946-13948, 14310, 14311,
14315-14317, 14679, 14680, 14684-14686, 15048, 15049, 15053-15055,
15417, 15418, 15422-15424, 15786, 15787, 15791-15793, 13203, 13204,
13208-13210, 13572, 13573, 13577-13579, 16155, 16156, 16160-16162,
16524, 16525, 16529-16531, 16893, 16894, 16898-16900, 17262, 17263,
17267-17269, 17631, 17632, 17636-17638, 18000, 18001, 18005-18007,
18369, 18370, 18374-18376, 18738, 18739, 18743-18745, 19107, 19108,
19112-19114, 19476, 19477, 19481-19483, 21363-21384, 21389-21410 or
a fragment or variant of any of these sequences (see also Table 3
and 4; opt1, 5, 6, 11). Additional information regarding each of
these suitable nucleic acid sequences encoding may also be derived
from the sequence listing, in particular from the details provided
therein under identifier <223>.
[0279] In preferred embodiment, the artificial RNA of the invention
comprises at least one coding sequence comprising a codon modified
nucleic acid sequence which is identical or at least 50%, 60%, 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to any one of the human codon usage
adapted nucleic acid sequence according to the SEQ ID NOs: 73, 488,
902, 1271, 1640, 2009, 2378, 2747, 3116, 3485, 3854, 4223, 4592,
4961, 5330, 5699, 6068, 6437, 6806, 7175, 7544, 7913, 11730, 12099,
12468, 12837, 13944, 14313, 14682, 15051, 15420, 15789, 13206,
13575, 16158, 16527, 16896, 17265, 17634, 18003, 18372, 18741,
19110, 19479 or a fragment or variant of any of these sequences
(see also Table 3 and 4; opt3). Additional information regarding
each of these suitable nucleic acid sequences encoding may also be
derived from the sequence listing, in particular from the details
provided therein under identifier <223>.
[0280] In particularly preferred embodiment, the artificial RNA of
the first aspect comprises at least one coding sequence comprising
a codon modified nucleic acid sequence which is identical or at
least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the C
maximized nucleic acid sequence according to the SEQ ID NOs: 72,
487, 901, 1270, 1639, 2008, 2377, 2746, 3115, 3484, 3853, 4222,
4591, 4960, 5329, 5698, 6067, 6436, 6805, 7174, 7543, 7912, 11729,
12098, 12467, 12836, 13943, 14312, 14681, 15050, 15419, 15788,
13205, 13574, 16157, 16526, 16895, 17264, 17633, 18002, 18371,
18740, 19109, 19478 or a fragment or variant of any of these
sequences (see also Table 3 and 4; opt2).
[0281] Additional information regarding each of these suitable
nucleic acid sequences encoding may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223>.
[0282] In preferred embodiment, the artificial RNA of the invention
comprises at least one coding sequence comprising a codon modified
nucleic acid sequence which is identical or at least 50%, 60%, 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to any one of the CAI maximized nucleic
acid sequence according to the SEQ ID NOs 74, 489, 903, 1272, 1641,
2010, 2379, 2748, 3117, 3486, 3855, 4224, 4593, 4962, 5331, 5700,
6069, 6438, 6807, 7176, 7545, 7914, 11731, 12100, 12469, 12838,
13945, 14314, 14683, 15052, 15421, 15790, 13207, 13576, 16159,
16528, 16897, 17266, 17635, 18004, 18373, 18742, 19111, 19480 or a
fragment or variant of any of these sequences (see also Table 3 and
4; opt4). Additional information regarding each of these suitable
nucleic acid sequences encoding may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223>.
[0283] In embodiments, the A/U content in the environment of the
ribosome binding site of the artificial nucleic acid, particularly
the artificial RNA of the invention may be increased compared to
the A/U content in the environment of the ribosome binding site of
its respective wild type nucleic acid. This modification (an
increased A/U content around the ribosome binding site) increases
the efficiency of ribosome binding to the nucleic acid, preferably
the RNA. An effective binding of the ribosomes to the ribosome
binding site in turn has the effect of an efficient translation of
the RNA. Accordingly, in a particularly preferred embodiment, the
artificial nucleic acid of the invention comprises a ribosome
binding site, also referred to as "Kozak sequence" identical to or
at least 80%, 85%, 90%, 95% identical to any one of the sequences
SEQ ID NOs: 41, 42, or fragments or variants thereof.
[0284] Preferred RSV polypeptide and nucleic acid coding sequences
("cds") are provided in Table 3A and 3B and Table 4A and 4B.
[0285] In Table 3A and 3B, Columns A to J represent specific
suitable constructs of the invention derived from RSV Fusion (F)
protein, wherein Column A provides suitable sequences for F0,
Column B provides suitable sequences for F-del, Column C provides
suitable sequences for F0_DSCav1, Column D provides suitable
sequences for F-del_DSCav1, Column E provides suitable sequences
for F_DSCav1_mut1, Column F provides suitable sequences for
F-del_DSCav1_mut1, Column G provides suitable sequences for
F_DSCav1_mut2, Column H provides suitable sequences for
F-del_DSCav1_mut2, Column I provides suitable sequences for
F_DSCav1_mut3, Column J provides suitable sequences for
F-del_DSCav1_mut3. The specific protein SEQ ID NOs as provided in
the sequence listing are in row 2 ("PRT"). The SEQ ID NOs of
corresponding wild type/non-modified coding sequences are provided
in row 3 ("wt"). The SEQ ID NOs of corresponding codon modified
coding sequences for each protein construct are provided in row 4
to row 10 ("opt1", "opt2", "opt3", "opt4", "opt5", "opt6",
"opt11"). In Table 3A, coding sequences derived from HRSV(A2) are
provided, in Table 3B coding sequences derived from
HRSV(Memphis-37) are provided. Further information is provided in
the <223> identifier for each of the respective SEQ ID NO in
the sequence listing.
TABLE-US-00003 TABLE 3A Preferred coding sequences encoding RSV F
(columns A-J), derived from HRSV(A2) A B C D E F G H I J PRT 68 483
898 1267 1636 2005 2374 2743 3112 3481 wt 69 484 opt1 70, 71, 485,
899, 1268, 1637, 2006, 2375, 2744, 3113, 3482, 21363 486, 900,
1269, 1638, 2007, 2376, 2745, 3114, 3483, 21364 21365 21366 21369
21370 21371 21372 21373 21374 opt2 72 487 901 1270 1639 2008 2377
2746 3115 3484 opt3 73 488 902 1271 1640 2009 2378 2747 3116 3485
opt4 74 489 903 1272 1641 2010 2379 2748 3117 3486 opt5 75 490 904
1273 1642 2011 2380 2749 3118 3487 opt6 76 491 905 1274 1643 2012
2381 2750 3119 3488 opt11 77 492 906 1275 1644 2013 2382 2751 3120
3489
TABLE-US-00004 TABLE 3B Preferred coding sequences encoding RSV F
(columns A-J), derived from HRSV(Memphis-37) A B C D E F G H I J
PRT 11726 12095 12464 12833 13940 14309 14678 15047 15416 15785 wt
11727 12096 opt1 11728, 12097, 12465, 12834, 13941, 14310, 14679,
15048, 15417, 15786, 21389 21390 12466, 12835, 13942, 14311, 14680,
15049, 15418, 15787, 21391 21392 21395 21396 21397 21398 21399
21400 opt2 11729 12098 12467 12836 13943 14312 14681 15050 15419
15788 opt3 11730 12099 12468 12837 13944 14313 14682 15051 15420
15789 opt4 11731 12100 12469 12838 13945 14314 14683 15052 15421
15790 opt5 11732 12101 12470 12839 13946 14315 14684 15053 15422
15791 opt6 11733 12102 12471 12840 13947 14316 14685 15054 15423
15792 opt11 11734 12103 12472 12841 13948 14317 14686 15055 15424
15793
[0286] In Table 4A and 4B, Columns K to V represent specific
suitable constructs of the invention derived from RSV Fusion (F)
protein, wherein Column K provides suitable sequences for
F_DSCav1_mut0, Column L provides suitable sequences for
F-del_DSCav1_mut0, Column M provides suitable sequences for
F_DSCav1_mut4, Column N provides suitable sequences for
F-del_DSCav1_mut4, Column O provides suitable sequences for
F_DSCav1_mut5, Column P provides suitable sequences for
F-del_DSCav1_mut5, Column Q provides suitable sequences for
F_DSCav1_mut6, Column R provides suitable sequences for
F-del_DSCav1_mut6, Column S provides suitable sequences for
F_DSCav1_mut7, Column T provides suitable sequences for
F-del_DSCav1_mut7, Column U provides suitable sequences for
F_DSCav1_mut8, Column V provides suitable sequences for
F-del_DSCav1_mut8. The specific protein SEQ ID NOs as provided in
the sequence listing are in row 2 ("PRT"). The SEQ ID NOs of
corresponding codon modified coding sequences for each protein
construct are provided in row 3 to row 9 ("opt1", "opt2", "opt3",
"opt4", "opt5", "opt6", "opt11"). In Table 4A, coding sequences
derived from HRSV(A2) are provided, in Table 4B coding sequences
derived from HRSV(Memphis-37) are provided. Further information is
provided in the <223> identifier for each of the respective
SEQ ID NO in the sequence listing.
TABLE-US-00005 TABLE 4A Preferred coding sequences encoding RSV F
(columns K-V), derived from HRSV(A2) K L M N O P Q R S T U V PRT
3850 4219 4588 4957 5326 5695 6064 6433 6802 7171 7540 7909 opt1
3851, 4220, 4589, 4958, 5327, 5696, 6065, 6434, 6803, 7172, 7541,
7910, 3852, 4221, 4590, 4959, 5328, 5697, 6066, 6435, 6804, 7173,
7542, 7911, 21367 21368 21375 21376 21377 21378 21379 21380 21381
21382 21383 21384 opt2 3853 4222 4591 4960 5329 5698 6067 6436 6805
7174 7543 7912 opt3 3854 4223 4592 4961 5330 5699 6068 6437 6806
7175 7544 7913 opt4 3855 4224 4593 4962 5331 5700 6069 6438 6807
7176 7545 7914 opt5 3856 4225 4594 4963 5332 5701 6070 6439 6808
7177 7546 7915 opt6 3857 4226 4595 4964 5333 5702 6071 6440 6809
7178 7547 7916 opt11 3858 4227 4596 4965 5334 5703 6072 6441 6810
7179 7548 7917
TABLE-US-00006 TABLE 4B Preferred coding sequences encoding RSV F
(columns K-V) derived from HRSV(Memphis-37) K L M N O P Q R S T U V
PRT 13202 13571 16154 16523 16892 17261 17630 17999 18368 18737
19106 19475 opt1 13203, 13572, 16155, 16524, 16893, 17262, 17631,
18000, 18369, 18738, 19107, 19476, 13204, 13573, 16156, 16525,
16894, 17263, 17632, 18001, 18370, 18739, 19108, 19477, 21393 21394
21401 21402 21403 21404 21405 21406 21407 21408 21409 21410 opt2
13205 13574 16157 16526 16895 17264 17633 18002 18371 18740 19109
19478 opt3 13206 13575 16158 16527 16896 17265 17634 18003 18372
18741 19110 19479 opt4 13207 13576 16159 16528 16897 17266 17635
18004 18373 18742 19111 19480 opt5 13208 13577 16160 16529 16898
17267 17636 18005 18374 18743 19112 19481 opt6 13209 13578 16161
16530 16899 17268 17637 18006 18375 18744 19113 19482 opt11 13210
13579 16162 16531 16900 17269 17638 18007 18376 18745 19114
19483
[0287] In embodiments, the artificial RNA of the first aspect is
monocistronic, bicistronic, or multicistronic.
[0288] In preferred embodiments, the artificial RNA of the
invention is monocistronic.
[0289] The term "monocistronic nucleic acid" or "monocistronic
nucleic acid" will be recognized and understood by the person of
ordinary skill in the art, and is for example intended to refer to
an artificial RNA that comprises only one coding sequences as
defined herein. The terms "bicistronic nucleic acid, multicistronic
nucleic acid" or "monocistronic RNA" as used herein will be
recognized and understood by the person of ordinary skill in the
art, and are for example intended to refer to an artificial RNA
that may have two (bicistronic) or even more (multicistronic)
coding sequences.
[0290] In embodiments, the artificial RNA of the invention is
monocistronic and the coding sequence of said monocistronic
artificial RNA encodes at least two different antigenic peptides or
proteins derived from RSV F as defined herein, or a fragment or
variant thereof. Accordingly, the at least one coding sequence of
the monocistronic artificial RNA may encode at least two, three,
four, five, six, seven, eight and more antigenic peptides or
proteins derived from a RSV, preferably a RSV F as defined herein
linked with or without an amino acid linker sequence, wherein said
linker sequence can comprise rigid linkers, flexible linkers,
cleavable linkers (e.g. self-cleaving peptides) as defined above,
or a combination thereof (herein referred to as
"multi-antigen-constructs/nucleic acid").
[0291] In embodiments, the artificial RNA of the invention is
bicistronic or multicistronic and comprises at least two coding
sequences, wherein the at least two coding sequences encode two or
more different antigenic peptides or proteins derived from RSV,
preferably RSV F as defined herein, or a fragment or variant of any
of these. Accordingly, the coding sequences in a bicistronic or
multicistronic artificial RNA suitably encodes distinct antigenic
proteins or peptides as defined herein or a fragment or variant
thereof. Preferably, the coding sequences in said bicistronic or
multicistronic artificial RNA may be separated by at least one IRES
(internal ribosomal entry site) sequence. Thus, the term "encoding
two or more antigenic peptides or proteins" may mean, without being
limited thereto, that the bicistronic or multicistronic artificial
RNA encodes e.g. at least two, three, four, five, six or more
(preferably different) antigenic peptides or proteins of different
RSV or their fragments or variants within the definitions provided
herein. Alternatively, the bicistronic or multicistronic artificial
RNA may encode e.g. at least two, three, four, five, six or more
(preferably different) antigenic peptides or proteins derived from
the same RSV or fragments or variants within the definitions
provided herein. In that context, suitable IRES sequences may be
selected from the list of nucleic acid sequences according to SEQ
ID NOs: 1566-1662 of the patent application WO2017/081082, or
fragments or variants of these sequences. In this context, the
disclosure of WO2017/081082 relating to IRES sequences is herewith
incorporated by reference.
[0292] It has to be understood that in the context of the
invention, certain combinations of coding sequences may be
generated by any combination of monocistronic, bicistronic and
multicistronic artificial nucleic acids and/or
multi-antigen-constructs/nucleic acid to obtain a nucleic acid
composition encoding multiple antigenic peptides or proteins as
defined herein.
[0293] Preferably, the artificial RNA comprising at least one
coding sequence as defined herein typically comprises a length of
about 50 to about 20000, or 500 to about 20000 nucleotides, or
about 500 to about 20000 nucleotides, or about 500 to about 10000
nucleotides, or of about 1000 to about 10000 nucleotides, or
preferably of about 1000 to about 5000 nucleotides, or even more
preferably of about 1000 to about 2500 nucleotides.
[0294] According to preferred embodiments, the artificial RNA of
the first aspect may be an mRNA, a self-replicating RNA, a circular
RNA, or a replicon RNA.
[0295] In embodiments, the artificial RNA is a circular RNA. As
used herein, "circular RNA" or "circRNAs" has to be understood as a
circular polynucleotide that can encode at least one antigenic
peptide or protein as defined herein. Accordingly, in preferred
embodiments, said circular RNA comprises at least one coding
sequence encoding at least one antigenic peptide or protein derived
from RSV or a fragment or variant thereof as defined herein. The
production of circRNAs can be performed using various methods
provided in the art. For example, U.S. Pat. No. 6,210,931 teaches a
method of synthesizing circRNAs by inserting DNA fragments into a
plasmid containing sequences having the capability of spontaneous
cleavage and self-circularization. U.S. Pat. No. 5,773,244 teaches
producing circRNAs by making a DNA construct encoding an RNA
cyclase ribozyme, expressing the DNA construct as an RNA, and then
allowing the RNA to self-splice, which produces a circRNA free from
intron in vitro. WO1992/001813 teaches a process of making single
strand circular nucleic acids by synthesizing a linear
polynucleotide, combining the linear nucleotide with a
complementary linking oligonucleotide under hybridization
conditions, and ligating the linear polynucleotide. The person
skilled in the art may also use methods provided in WO2015/034925
or WO2016/011222 to produce circular RNA. Accordingly, methods for
producing circular RNA as provided in U.S. Pat. Nos. 6,210,931,
5,773,244, WO1992/001813, WO2015/034925 and WO2016/011222 are
incorporated herewith by reference.
[0296] In embodiments, the artificial RNA is a replicon RNA. The
term "replicon RNA" will be recognized and understood by the person
of ordinary skill in the art, and are for example intended to be
optimized self-replicating artificial RNA constructs. Such
constructs include replication elements (replicase) derived from
alphaviruses and the substitution of the structural virus proteins
with the artificial nucleic acid of interest (in the context of the
invention, an artificial nucleic acid comprising at least one
coding sequence encoding at least one antigenic peptide or protein
derived from RSV. Alternatively, the replicase may be provided on
an independent construct comprising a replicase RNA sequence
derived from e.g. Semliki forest virus (SFV), Sindbis virus (SIN),
Venezuelan equine Encephalitis virus (VEE), Ross-River virus (RRV),
or other viruses belonging to the alphavirus family. Downstream of
the replicase lies a sub-genomic promoter that controls replication
of the artificial nucleic acid of the invention, i.e. an artificial
nucleic acid comprising at least one coding sequence encoding at
least one antigenic peptide or protein derived from RSV.
[0297] In preferred embodiments the artificial RNA of the first
aspect is an mRNA.
[0298] The terms "RNA" and "mRNA" will be recognized and understood
by the person of ordinary skill in the art, and are for example
intended to be a ribonucleic acid molecule, i.e. a polymer
consisting of nucleotides. These nucleotides are usually
adenosine-monophosphate, uridine-monophosphate,
guanosine-monophosphate and cytidine-monophosphate monomers which
are connected to each other along a so-called backbone. The
backbone is formed by phosphodiester bonds between the sugar, i.e.
ribose, of a first and a phosphate moiety of a second, adjacent
monomer. The specific succession of the monomers is called the
RNA-sequence. The mRNA (messenger RNA) usually provides the
nucleotide sequence that may be translated into an amino-acid
sequence of a particular peptide or protein.
[0299] The artificial RNA, preferably the mRNA of the invention may
be prepared using any method known in the art, including chemical
synthesis such as e.g. solid phase RNA synthesis, as well as in
vitro methods, such as RNA in vitro transcription reactions.
[0300] In a preferred embodiment, the artificial RNA, preferably
the mRNA is obtained by RNA in vitro transcription.
[0301] Accordingly, the RNA of the invention is preferably an in
vitro transcribed RNA.
[0302] The terms "RNA in vitro transcription" or "in vitro
transcription" relate to a process wherein RNA is synthesized in a
cell-free system (in vitro). RNA may be obtained by DNA-dependent
in vitro transcription of an appropriate DNA template, which
according to the present invention is a linearized plasmid DNA
template or a PCR-amplified DNA template. The promoter for
controlling RNA in vitro transcription can be any promoter for any
DNA-dependent RNA polymerase. Particular examples of DNA-dependent
RNA polymerases are the T7, T3, SP6, or Syn5 RNA polymerases. In a
preferred embodiment of the present invention the DNA template is
linearized with a suitable restriction enzyme, before it is
subjected to RNA in vitro transcription.
[0303] Reagents used in RNA in vitro transcription typically
include: a DNA template (linearized plasmid DNA or PCR product)
with a promoter sequence that has a high binding affinity for its
respective RNA polymerase such as bacteriophage-encoded RNA
polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates
(NTPs) for the four bases (adenine, cytosine, guanine and uracil);
optionally, a cap analogue as defined herein (e.g. m7G(5')ppp(5')G
(m7G, m7G(5')ppp(5')(2'OMeG)pG or m7G(5')ppp(5')(2'OMeA)pG));
optionally, further modified nucleotides as defined herein; a
DNA-dependent RNA polymerase capable of binding to the promoter
sequence within the DNA template (e.g. T7, T3, SP6, or Syn5 RNA
polymerase); optionally, a ribonuclease (RNase) inhibitor to
inactivate any potentially contaminating RNase; optionally, a
pyrophosphatase to degrade pyrophosphate, which may inhibit RNA in
vitro transcription; MgCl2, which supplies Mg2+ ions as a co-factor
for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable
pH value, which can also contain antioxidants (e.g. DTT), and/or
polyamines such as spermidine at optimal concentrations, e.g. a
buffer system comprising TRIS-Citrate as disclosed in
WO2017/109161.
[0304] In embodiments, the nucleotide mixture used in RNA in vitro
transcription may additionally contain modified nucleotides as
defined herein. In that context, preferred modified nucleotides
comprise pseudouridine (.psi.), N1-methylpseudouridine (m1.psi.),
5-methylcytosine, and/or 5-methoxyuridine.
[0305] In preferred embodiments, the nucleotide mixture (i.e. the
fraction of each nucleotide in the mixture) used for RNA in vitro
transcription reactions may be optimized for the given RNA
sequence, preferably as described WO2015/188933.
[0306] In embodiment where more than one different artificial RNA
as defined herein has to be produced, e.g. where 2, 3, 4, 5, 6, 7,
8, 9, 10 or even more different artificial RNAs have to be produced
(e.g. encoding different RSV F antigens, or e.g. a combination of
RSV F and RSV G; see second aspect), procedures as described in
WO2017/109134 may be suitably used.
[0307] In the context of RNA vaccine production, it may be required
to provide GMP-grade RNA. GMP-grade RNA may be produced using a
manufacturing process approved by regulatory authorities.
Accordingly, in a particularly preferred embodiment, RNA production
is performed under current good manufacturing practice (GMP),
implementing various quality control steps on DNA and RNA level,
preferably according to WO2016/180430. In preferred embodiments,
the RNA of the invention is a GMP-grade RNA, particularly a
GMP-grade mRNA.
[0308] The obtained RNA products are preferably purified using
PureMessenger.RTM. (CureVac, Tubingen, Germany; RP-HPLC according
to WO2008/077592) and/or tangential flow filtration (as described
in WO2016/193206).
[0309] In a further preferred embodiment, the RNA, particularly the
purified RNA, is lyophilized according to WO2016/165831 or
WO2011/069586 to yield a temperature stable dried artificial RNA
(powder) as defined herein. The RNA of the invention, particularly
the purified RNA may also be dried using spray-drying or
spray-freeze drying according to WO2016/184575 or WO2016184576 to
yield a temperature stable RNA (powder) as defined herein.
Accordingly, in the context of manufacturing and purifying RNA, the
disclosures of WO2017/109161, WO2015/188933, WO2016/180430,
WO2008/077592, WO2016/193206, WO2016/165831, WO2011/069586,
WO2016/184575, and WO2016/184576 are incorporated herewith by
reference.
[0310] Accordingly, in preferred embodiments, the RNA is a dried
RNA, particularly a dried mRNA.
[0311] The term "dried RNA" as used herein has to be understood as
RNA that has been lyophilized, or spray-dried, or spray-freeze
dried as defined above to obtain a temperature stable dried RNA
(powder).
[0312] In preferred embodiments, the artificial RNA of the
invention is a purified RNA, particularly purified mRNA.
[0313] The term "purified RNA" or "purified mRNA" as used herein
has to be understood as RNA which has a higher purity after certain
purification steps (e.g. HPLC, TFF, Oligo d(T) purification,
precipitation steps) than the starting material (e.g. in vitro
transcribed RNA). Typical impurities that are essentially not
present in purified RNA comprise peptides or proteins (e.g. enzymes
derived from DNA dependent RNA in vitro transcription, e.g. RNA
polymerases, RNases, pyrophosphatase, restriction endonuclease,
DNase), spermidine, BSA, abortive RNA sequences, RNA fragments
(short double stranded RNA fragments, abortive sequences etc.),
free nucleotides (modified nucleotides, conventional NTPs, cap
analogue), template DNA fragments, buffer components (HEPES, TRIS,
MgCl2) etc. Other potential impurities that may be derived from
e.g. fermentation procedures comprise bacterial impurities
(bioburden, bacterial DNA) or impurities derived from purification
procedures (organic solvents etc.). Accordingly, it is desirable in
this regard for the "degree of RNA purity" to be as close as
possible to 100%. It is also desirable for the degree of RNA purity
that the amount of full-length RNA transcripts is as close as
possible to 100%. Accordingly "purified RNA" as used herein has a
degree of purity of more than 75%, 80%, 85%, very particularly 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favorably 99% or
more. The degree of purity may for example be determined by an
analytical HPLC, wherein the percentages provided above correspond
to the ratio between the area of the peak for the target RNA and
the total area of all peaks representing the by-products.
Alternatively, the degree of purity may for example be determined
by an analytical agarose gel electrophoresis or capillary gel
electrophoresis.
[0314] It has to be understood that "dried RNA" as defined herein
and "purified RNA" as defined herein or "GMP-grade mRNA" as defined
herein may have superior stability characteristics (in vitro, in
vivo) and improved efficiency (e.g. better translatability of the
mRNA in vivo) and are therefore particularly suitable in the
context of the invention. Moreover, "dried RNA" as defined herein
and "purified RNA" as defined herein or "GMP-grade mRNA" may be
particularly suitable for medical use as defined herein.
[0315] The artificial RNA may suitably be modified by the addition
of a 5'-cap structure, which preferably stabilizes the nucleic acid
as described herein. Accordingly, in preferred embodiments, the
artificial RNA of the first aspect comprises a 5'-cap structure,
preferably m7G, cap0 (e.g. m7G(5')ppp(5')G), cap1 (e.g.
m7G(5')ppp(5')(2'OMeG) or m7G(5')ppp(5')(2'OMeA)), cap2, a modified
cap0, or a modified cap1 structure (generated using a cap analogue
as defined below).
[0316] The term "5'-cap structure" as used herein will be
recognized and understood by the person of ordinary skill in the
art, and is for example intended to refer to a modified nucleotide
(cap analogue), particularly a guanine nucleotide, added to the
5'-end of an RNA molecule, e.g. an mRNA molecule. Preferably, the
5'-cap is added using a 5-5-triphosphate linkage (also named
m7GpppN). Further examples of 5'-cap structures include glyceryl,
inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide,
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide,
carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,
L-nucleotides, alpha-nucleotide, modified base nucleotide,
threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide,
acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl
nucleotide, 3'-3-inverted nucleotide moiety, 3'-3'-inverted abasic
moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic
moiety, 1,4-butanediol phosphate, 3'-phosphoramidate,
hexylphosphate, aminohexyl phosphate, 3'-phosphate,
3'phosphorothioate, phosphorodithioate, or bridging or non-bridging
methylphosphonate moiety.
[0317] Further 5'-cap structures which may be suitable in the
context of the present invention are cap1 (additional methylation
of the ribose of the adjacent nucleotide of m7GpppN), cap2
(additional methylation of the ribose of the 2nd nucleotide
downstream of the m7GpppN), cap3 (additional methylation of the
ribose of the 3rd nucleotide downstream of the m7GpppN), cap4
(additional methylation of the ribose of the 4th nucleotide
downstream of the m7GpppN), ARCA (anti-reverse cap analogue),
modified ARCA (e.g. phosphothioate modified ARCA), inosine,
N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine,
8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and
2-azido-guanosine.
[0318] A 5'-cap (cap0 or cap1) structure may also be formed in
chemical RNA synthesis or, preferably, RNA in vitro transcription
(co-transcriptional capping) using cap analogues.
[0319] The term "cap analogue" as used herein will be recognized
and understood by the person of ordinary skill in the art, and is
for example intended to refer to a non-polymerizable di-nucleotide
that has cap functionality in that it facilitates translation or
localization, and/or prevents degradation of a nucleic acid
molecule, particularly of an RNA molecule, when incorporated at the
5'-end of the nucleic acid molecule. Non-polymerizable means that
the cap analogue will be incorporated only at the 5'-terminus
because it does not have a 5' triphosphate and therefore cannot be
extended in the 3'-direction by a template-dependent polymerase,
particularly, by template-dependent RNA polymerase. Examples of cap
analogues include, but are not limited to, a chemical structure
selected from the group consisting of m7GpppG, m7GpppA, m7GpppC;
unmethylated cap analogues (e.g. GpppG); dimethylated cap analogue
(e.g. m2,7GpppG), trimethylated cap analogue (e.g. m2,2,7GpppG),
dimethylated symmetrical cap analogues (e.g. m7Gpppm7G), or anti
reverse cap analogues (e.g. ARCA; m7,2'OmeGpppG, m7,2'dGpppG,
m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives).
Further cap analogues have been described previously
(WO2008/016473, WO2008/157688, WO2009/149253, WO2011/015347, and
WO2013/059475). Further suitable cap analogons in that context are
described in WO2017/066793, WO2017/066781, WO2017/066791,
WO2017/066789, WO2017/053297, WO2017/066782, WO2018075827 and
WO2017/066797 wherein the disclosures referring to cap analogues
are incorporated herewith by reference.
[0320] The 5'-cap structure may suitably be added
co-transcriptionally using cap-analogues as defined herein in an
RNA in vitro transcription reaction as defined herein.
[0321] In preferred embodiments, a modified cap1 structure is
generated using a cap analogue as disclosed in WO2017/053297,
WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789,
WO2017/066782, WO2018075827 and WO2017/066797. In particular, any
cap structures derivable from the structure disclosed in claim 1-5
of WO2017/053297 may be suitably used to co-transcriptionally
generate a modified cap1 structure. Further, any cap structures
derivable from the structure defined in claim 1 or claim 21 of
WO2018075827 may be suitably used to co-transcriptionally generate
a modified cap1 structure.
[0322] Preferred cap-analogues are the di-nucleotide cap analogues
m7G(5')ppp(5')G (m7G) or 3 O-Me-m7G(5')ppp(5')G to
co-transcriptionally generate cap0 structures. Particularly
preferred cap-analogues are the tri-nucleotide cap analogues
m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG to
co-transcriptionally generate cap1 structures.
[0323] In that context, it is preferred that the RNA of the
invention comprises a Cap1 structure as defined above, which
preferably result in an increased protein expression through e.g.
high capping efficiencies and increased translation efficiencies.
Further suitably, the RNA of the invention comprising a Cap1
structure displays a decreased stimulation of the innate immune
system as compared to Cap0 constructs of the same nucleic acid
sequence. The person of ordinary skill knows how to determine
translation efficiencies, capping degree, and immune
stimulation.
[0324] In a particularly preferred embodiment, the artificial RNA
of the first aspect of the invention comprises a cap1 structure,
wherein said cap1 structure may be formed enzymatically or
co-transcriptionally (e.g. using m7G(5')ppp(5')(2'OMeA)pG or
m7G(5')ppp(5')(2'OMeG)pG analogues).
[0325] In preferred embodiments, the artificial RNA of the first
aspect comprises an m7G(5')ppp(5')(2'OMeA)pG cap structure. In such
embodiments, the coding RNA comprises a 5' terminal m7G cap, and an
additional methylation of the ribose of the adjacent nucleotide of
m7GpppN, in that case, a 2'O methylated adenosine.
[0326] In other preferred embodiments, the artificial RNA of the
first aspect comprises an m7G(5')ppp(5')(2'OMeG)pG cap structure.
In such embodiments, the coding RNA comprises a 5' terminal m7G
cap, and an additional methylation of the ribose of the adjacent
nucleotide, in that case, a 2'O methylated guanosine.
[0327] Accordingly, whenever reference is made to suitable RNA or
mRNA sequences in the context of the invention, the first
nucleotide of said RNA or mRNA sequence, that is the nucleotide
downstream of the m7G(5')ppp structure, may be a 2'O methylated
guanosine or a 2'O methylated adenosine.
[0328] Accordingly, in other embodiments, the artificial RNA of the
invention may comprise a 5'-cap sequence element according to SEQ
ID NOs: 43 or 21321, or a fragment or variant thereof.
[0329] In other embodiments, the 5'-cap structure is added via
enzymatic capping using capping enzymes (e.g. vaccinia virus
capping enzymes, commercially available capping kits) to generate
cap or cap1 or cap2 structures. In other embodiments, the 5'-cap
structure (cap0, cap1) is added via enzymatic capping using
immobilized capping enzymes, e.g. using a capping reactor
(WO2016/193226).
[0330] In preferred embodiments, the artificial RNA of the
invention comprises at least one poly(A) sequence, preferably
comprising 30 to 150 adenosine nucleotides.
[0331] In preferred embodiments, the poly(A) sequence, suitable
located at the 3' terminus, comprises 10 to 500 adenosine
nucleotides, 10 to 200 adenosine nucleotides, 40 to 200 adenosine
nucleotides or 40 to 150 adenosine nucleotides. In a particularly
preferred embodiment, the poly(A) sequence comprises about 64
adenosine nucleotides. In further particularly preferred
embodiments, the poly(A) sequence comprises about 75 adenosine
nucleotides. In further particularly preferred embodiments, the
poly(A) sequence comprises about 100 adenosine nucleotides.
[0332] The terms "poly(A) sequence", "poly(A) tail" or "3'-poly(A)
tail" as used herein will be recognized and understood by the
person of ordinary skill in the art, and are for example intended
to be a sequence of adenosine nucleotides, typically located at the
3'-end of an RNA, of up to about 1000 adenosine nucleotides.
Preferably, said poly(A) sequence is essentially homopolymeric,
e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides has
essentially the length of 100 nucleotides. In other embodiments,
the poly(A) sequence may be interrupted by at least one nucleotide
different from an adenosine nucleotide, e.g. a poly(A) sequence of
e.g. 100 adenosine nucleotides may have a length of more than 100
nucleotides (comprising 100 adenosine nucleotides and in addition
said at least one nucleotide different from an adenosine
nucleotide).
[0333] In the context of the present invention, a poly(A) sequence
may be located within an mRNA or any other nucleic acid molecule,
such as, e.g., in a DNA vector, for example, in a vector serving as
template for the generation of an RNA, preferably an mRNA, e.g., by
transcription said DNA vector.
[0334] Preferably, the poly(A) sequence of the artificial RNA is
obtained from a DNA template during RNA in vitro transcription. In
other embodiments, the poly(A) sequence is obtained in vitro by
common methods of chemical synthesis without being necessarily
transcribed from a DNA template. In other embodiments, poly(A)
sequences are generated by enzymatic polyadenylation of the RNA
(after RNA in vitro transcription) using commercially available
polyadenylation kits and corresponding protocols known in the art,
or alternatively, by using immobilized poly(A)polymerases e.g.
using a polyadenylation reactor (as described in
WO2016/174271).
[0335] In embodiments, the artificial RNA of the invention may
contain a poly(A) sequence derived from a vector and may comprise
at least one additional poly(A) sequence generated by enzymatic
polyadenylation, e.g. as described in WO2016/091391.
[0336] In preferred embodiments, the artificial RNA of the
invention comprises at least one poly(C) sequence, preferably
comprising 10 to 40 cytosine nucleotides.
[0337] In preferred embodiments, the poly(C) sequence, suitable
located at the 3' terminus, comprises 10 to 200 cytosine
nucleotides, 10 to 100 cytosine nucleotides, 20 to 70 cytosine
nucleotides, 20 to 60 cytosine nucleotides, or 10 to 40 cytosine
nucleotides. In a particularly preferred embodiment, the poly(C)
sequence comprises about cytosine nucleotides.
[0338] The term "poly(C) sequence" as used herein will be
recognized and understood by the person of ordinary skill in the
art, and are for example intended to be a sequence of cytosine
nucleotides, typically located at the 3'-end of an RNA, of up to
about 200 cytosine nucleotides. In the context of the present
invention, a poly(C) sequence may be located within an mRNA or any
other nucleic acid molecule, such as, e.g., in a DNA vector, for
example, in a vector serving as template for the generation of an
RNA, preferably an mRNA, e.g., by transcription of the vector.
[0339] Preferably, the poly(C) sequence in the RNA sequence of the
present invention is derived from a DNA template by RNA in vitro
transcription. In other embodiments, the poly(C) sequence is
obtained in vitro by common methods of chemical synthesis without
being necessarily transcribed from a DNA template.
[0340] In other embodiments, the artificial RNA of the invention
does not comprises a poly(C) sequence as defined herein.
[0341] In preferred embodiments, the artificial RNA of the first
aspect comprises at least one histone stem-loop.
[0342] The term "histone stem-loop" as used herein will be
recognized and understood by the person of ordinary skill in the
art, and are for example intended to refer to nucleic acid
sequences that are predominantly found in histone mRNAs. Exemplary
histone stem-loop sequences are described in Lopez et al. (Davila
Lopez et al, (2008), RNA, 14(1)). The stem-loops in histone
pre-mRNAs are typically followed by a purine-rich sequence known as
the histone downstream element (HDE). These pre-mRNAs are processed
in the nucleus by a single endonucleolytic cleavage approximately 5
nucleotides downstream of the stem-loop, catalysed by the U7 snRNP
through base pairing of the U7 snRNA with the HDE.
[0343] Histone stem-loop sequences/structures may suitably be
selected from histone stem-loop sequences as disclosed in
WO2012/019780, the disclosure relating to histone stem-loop
sequences/structures incorporated herewith by reference. A histone
stem-loop sequence that may be used within the present invention
may preferably be derived from formulae (I) or (II) of the patent
application WO2012/019780. According to a further preferred
embodiment the RNA as defined herein may comprise at least one
histone stem-loop sequence derived from at least one of the
specific formulae (Ia) or (IIa) of the patent application
WO2012/019780.
[0344] In particularly preferred embodiment, the artificial RNA of
the invention comprises at least one histone stem-loop, wherein
said histone stem-loop comprises a nucleic acid sequence according
to SEQ ID NO: 39 or 40 or a fragments or variant thereof.
[0345] In other embodiments, the artificial RNA of the first aspect
does not comprises a histone stem-loop as defined herein.
[0346] In further embodiments, the artificial RNA of the invention
comprises a 3'-terminal sequence element. Said 3'-terminal sequence
element has to be understood as a sequence element comprising a
poly(A)sequence and a histone-stem-loop sequence, wherein said
sequence element is located at the 3' terminus of the artificial
RNA of the invention.
[0347] In other embodiments, the artificial RNA of the invention
may comprise a 3'-terminal sequence element according to SEQ ID
NOs: 44-63 or 21322-21328 or a fragment or variant thereof.
[0348] In preferred embodiments, the artificial RNA of the
invention comprises at least one pseudouridine (.psi.) modified
coding sequence.
[0349] Accordingly, in preferred embodiments, the artificial RNA of
the invention, or the at least one coding sequence, comprises a
nucleic acid sequence wherein at least one or more than one,
preferably wherein all uracil nucleotides are replaced by
pseudouridine (.psi.) nucleotides.
[0350] In further preferred embodiments, the artificial RNA of the
invention comprises at least one N1-methylpseudouridine (m1.psi.)
modified coding sequence.
[0351] Accordingly, in preferred embodiments, the artificial RNA of
the invention, or the at least one coding sequence, comprises a
nucleic acid sequence wherein at least one or more than one,
preferably wherein all uracil nucleotides are replaced by
N1-methylpseudouridine (m1P) nucleotides
[0352] In preferred embodiments of the first aspect, the artificial
RNA, preferably mRNA comprises preferably in 5'- to 3-direction the
following elements a)-i): [0353] a) 5'-cap structure, preferably as
specified herein; [0354] b) optionally, 5'-UTR as specified herein,
preferably at least one selected from SEQ ID NOs: 1-22; [0355] c) a
ribosome binding site, preferably as specified herein [0356] d) at
least one coding sequence as specified herein, preferably as
specified in Table 3 and Table 4; [0357] e) 3'-UTR as specified
herein, preferably at least one selected from SEQ ID NOs: 23-38;
[0358] f) optionally, a poly(A) sequence, preferably as specified
herein; [0359] g) optionally, a poly(C) sequence, preferably as
specified herein; [0360] h) optionally, a histone stem-loop,
preferably as specified herein; [0361] i) optionally, a 3'-terminal
sequence element as specified herein, preferably according to
according to SEQ ID NOs: 44-63, or 21322-21328; and [0362] wherein
optionally at least one or more than one, preferably wherein all
uracil nucleotides are replaced by pseudouridine (L) nucleotides or
N1-methylpseudouridine (m1.psi.) nucleotides.
[0363] In further preferred embodiments of the first aspect, the
artificial RNA, preferably mRNA comprises the following elements
preferably in 5'- to 3-direction: [0364] a) 5'-cap structure,
preferably as specified herein, most preferably a Cap1 structure;
[0365] b) a 3'-UTR and a 5'-UTR element according to a-1, a-4, c-1,
e-4, g-2, i-2, or i-3 as specified herein; [0366] c) a ribosome
binding site, preferably as specified herein; [0367] d) at least
one coding sequence as specified herein, wherein said coding
sequence is located between said 5'-UTR and said 3'-UTR, preferably
downstream of said 5'-UTR and upstream of said 3'-UTR, wherein the
coding sequence is preferably selected from any one specified in
Table 3 and Table 4; [0368] e) optionally, a poly(A) sequence,
preferably as specified herein; [0369] f) optionally, poly(C)
sequence, preferably as specified herein; [0370] g) optionally,
histone stem-loop, preferably as specified herein; [0371] h)
optionally, a 3'-terminal sequence element as specified herein,
preferably according to according to SEQ ID NOs: 44-63,
21322-21328, and [0372] wherein optionally at least one or more
than one, preferably wherein all uracil nucleotides are replaced by
pseudouridine (.psi.) nucleotides or N1-methylpseudouridine
(m1.psi.) nucleotides.
[0373] In further preferred embodiments of the first aspect, the
artificial RNA, preferably mRNA comprises the following elements
preferably in 5'- to 3-direction: [0374] a) 5'-cap structure,
preferably as specified herein, most preferably a Cap1 structure;
[0375] b) a 3'-UTR and a 5'-UTR element according to a-1 or i-3 as
specified herein; [0376] c) a ribosome binding site, preferably as
specified herein [0377] d) at least one coding sequence as
specified herein, wherein said coding sequence is located between
said 5'-UTR and said 3'-UTR, preferably downstream of said 5'-UTR
and upstream of said 3'-UTR, wherein the coding sequence is
preferably selected from any one specified in Table 3 and Table 4;
[0378] e) optionally, a histone stem-loop, preferably as specified
herein; [0379] f) a poly(A) sequence, preferably comprising about
100 adenosine nucleotides; [0380] g) optionally, a 3'-terminal
sequence element as specified herein, preferably according to
according to SEQ ID NOs: 21322-21328, and [0381] wherein optionally
at least one or more than one, preferably wherein all uracil
nucleotides are replaced by pseudouridine (.psi.) nucleotides or
N1-methylpseudouridine (m1.gamma.) nucleotides.
[0382] In further preferred embodiments of the first aspect, the
artificial RNA, preferably mRNA comprises the following elements in
5'- to 3'-direction: [0383] a) 5'-cap structure, preferably as
specified herein, most preferably a Cap1 structure; [0384] b) a
3'-UTR and a 5'-UTR element according to a-1, a-4, c-1, e-4, g-2,
i-2, or i-3 as specified herein; [0385] c) a ribosome binding site,
preferably as specified herein; [0386] d) at least one coding
sequence as specified herein, wherein said coding region is located
between said 5'-UTR and said 3'-UTR, preferably downstream of said
5'-UTR and upstream of said 3'-UTR, wherein the coding sequence is
preferably selected from any one of SEQ ID NOs: 69-77, 484-492,
899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751,
3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965,
5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179,
7541-7548, 7910-7917, 11727-11734, 12096-12103, 12465-12472,
12834-12841, 13941-13948, 14310-14317, 14679-14686, 15048-15055,
15417-15424, 15786-15793, 13203-13210, 13572-13579, 16155-16162,
16524-16531, 16893-16900, 17262-17269, 17631-17638, 18000-18007,
18369-18376, 18738-18745, 19107-19114, 19476-19483, 21363-21384,
21389-21410 (or fragments or variants thereof); [0387] e) a poly(A)
sequence comprising about 64 adenosine; [0388] f) a poly(C)
sequence comprising about 30 cysteines; [0389] g) a histone
stem-loop according to SEQ ID NO: 39 or 40, and [0390] wherein
optionally at least one or more than one, preferably wherein all
uracil nucleotides are replaced by pseudouridine (.psi.)
nucleotides or N1-methylpseudouridine (m1.psi.) nucleotides.
[0391] In further preferred embodiments of the first aspect, the
artificial RNA, preferably mRNA comprises the following elements in
5'- to 3'-direction: [0392] a) 5'-cap structure, preferably as
specified herein, most preferably a Cap1 structure; [0393] b) a
3'-UTR and a 5'-UTR element according to a-1, a-4, c-1, e-4, g-2,
i-2, or i-3 as specified herein; [0394] c) a ribosome binding site,
preferably as specified herein; [0395] d) at least one coding
sequence as specified herein, wherein said coding region is located
between said 5'-UTR and said 3'-UTR, preferably downstream of said
5'-UTR and upstream of said 3'-UTR, wherein the coding sequence is
preferably selected from any one of SEQ ID NOs: 69-77, 484-492,
899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751,
3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965,
5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179,
7541-7548, 7910-7917, 11727-11734, 12096-12103, 12465-12472,
12834-12841, 13941-13948, 14310-14317, 14679-14686, 15048-15055,
15417-15424, 15786-15793, 13203-13210, 13572-13579, 16155-16162,
16524-16531, 16893-16900, 17262-17269, 17631-17638, 18000-18007,
18369-18376, 18738-18745, 19107-19114, 19476-19483, 21363-21384,
21389-21410 (or fragments or variants thereof); [0396] e) a poly(A)
sequence comprising about 64 adenosine; [0397] f) a histone
stem-loop according to SEQ ID NO: 39 or 40, and [0398] wherein
optionally at least one or more than one, preferably wherein all
uracil nucleotides are replaced by pseudouridine (.psi.)
nucleotides or N1-methylpseudouridine (m1.psi.) nucleotides.
[0399] Preferred RSV polypeptide, nucleic acid, and mRNA sequences
are provided in Table 5A, 5B and Table 6A, 6B.
[0400] In Table 5A and 5B, the protein designs are indicated in row
1. Therein, Columns A to J represent specific suitable constructs
of the invention derived from RSV Fusion (F) protein, wherein
Column A provides suitable sequences for F0, Column B provides
suitable sequences for F-del, Column C provides suitable sequences
for F0_DSCav1, Column D provides suitable sequences for
F-del_DSCav1, Column E provides suitable sequences for
F_DSCav1_mut1, Column F provides suitable sequences for
F-del_DSCav1_mut1, Column G provides suitable sequences for
F_DSCav1_mut2, Column H provides suitable sequences for
F-del_DSCav1_mut2, Column I provides suitable sequences for
F_DSCav1_mut3, Column J provides suitable sequences for
F-del_DSCav1_mut3.
[0401] The protein designs are indicated in row 1 (Columns A to J),
the specific protein SEQ ID NOs as provided in the sequence listing
are in row 2 ("protein"). The SEQ ID NOs of corresponding coding
sequences for each protein construct are provided in row 3 ("cds",
compare with Table 3 for different cds optimizations). Further
information e.g. regarding the type of codon modified coding
sequence (opt1, opt2, opt3, opt4, opt5, opt6, opt11 etc.) is
provided in the <223> identifier of the respective SEQ ID NO
in the sequence listing and in Table 3. The SEQ ID NOs of
corresponding mRNA constructs comprising said coding sequences and
suitable 3'-UTRs and 5'-UTRs according to the invention are
provided in rows 4 to 47 (mRNA designs a-1 to i-3 as specified
herein). In Table 5A, mRNA sequences derived from HRSV(A2) are
provided, in Table 5B mRNA sequences derived from HRSV(Memphis-37)
are provided. Further information e.g. regarding the type of coding
sequence (wt, opt1, opt2, opt3, opt4, opt5, opt6, opt11 etc.)
comprised in the mRNA constructs is provided in the <223>
identifier of the respective SEQ ID NO in the sequence listing.
TABLE-US-00007 TABLE 5A Preferred mRNA constructs encoding RSV F
(columns A-J), derived from HRSV(A2) 1 A B C D E F G H I J 2
Protein 68 483 898 1267 1636 2005 2374 2743 3112 3481 3 cds 69-77,
484- 899- 1268- 1637- 2006- 2375- 2744- 3113- 3482- 21363 492, 906,
1275, 1644, 2013, 2382, 2751, 3120, 3489, 21364 21365 21366 21369
21370 21371 21372 21373 21374 4 mRNA 78-86, 493- 907- 1276- 1645-
2014- 2383- 2752- 3121- 3490- Design a-1 21415- 501, 914, 1283,
1652, 2021, 2390, 2759, 3128, 3497, 21417, 21418- 21421- 21424-
21433- 21436- 21439- 21442- 21445- 21448- 21561- 21420, 21423,
21426, 21435, 21438, 21441, 21444, 21447, 21450, 21563 21564-
21567- 21570- 21579- 21582- 21585- 21588- 21591- 21594- 21566 21569
21572 21581 21584 21587 21590 21593 21596 5 mRNA 87-95 502- 915-
1284- 1653- 2022- 2391- 2760- 3129- 3498- Design a-2 510 922 1291
1660 2029 2398 2767 3136 3505 6 mRNA 96-104 511- 923- 1292- 1661-
2030- 2399- 2768- 3137- 3506- Design a-3 519 930 1299 1668 2037
2406 2775 3144 3513 7 mRNA 105-113 520- 931- 1300- 1669- 2038-
2407- 2776- 3145- 3514- Design a-4 528 938 1307 1676 2045 2414 2783
3152 3521 8 mRNA 114-122 529- 939- 1308- 1677- 2046- 2415- 2784-
3153- 3522- Design a-5 537 946 1315 1684 2053 2422 2791 3160 3529 9
mRNA 123-131 538- 947- 1316- 1685- 2054- 2423- 2792- 3161- 3530-
Design b-1 546 954 1323 1692 2061 2430 2799 3168 3537 10 mRNA
132-140 547- 955- 1324- 1693- 2062- 2431- 2800- 3169- 3538- Design
b-2 555 962 1331 1700 2069 2438 2807 3176 3545 11 mRNA 141-149 556-
963- 1332- 1701- 2070- 2439- 2808- 3177- 3546- Design b-3 564 970
1339 1708 2077 2446 2815 3184 3553 12 mRNA 150-158 565- 971- 1340-
1709- 2078- 2447- 2816- 3185- 3554- Design b-4 573 978 1347 1716
2085 2454 2823 3192 3561 13 mRNA 159-167 574- 979- 1348- 1717-
2086- 2455- 2824- 3193- 3562- Design b-5 582 986 1355 1724 2093
2462 2831 3200 3569 14 mRNA 168-176 583- 987- 1356- 1725- 2094-
2463- 2832- 3201- 3570- Design c-1 591 994 1363 1732 2101 2470 2839
3208 3577 15 mRNA 177-185 592- 995- 1364- 1733- 2102- 2471- 2840-
3209- 3578- Design c-2 600 1002 1371 1740 2109 2478 2847 3216 3585
16 mRNA 186-194 601- 1003- 1372- 1741- 2110- 2479- 2848- 3217-
3586- Design c-3 609 1010 1379 1748 2117 2486 2855 3224 3593 17
mRNA 195-203 610- 1011- 1380- 1749- 2118- 2487- 2856- 3225- 3594-
Design c-4 618 1018 1387 1756 2125 2494 2863 3232 3601 18 mRNA
204-212 619- 1019- 1388- 1757- 2126- 2495- 2864- 3233- 3602- Design
c-5 627 1026 1395 1764 2133 2502 2871 3240 3609 19 mRNA 213-221
628- 1027- 1396- 1765- 2134- 2503- 2872- 3241- 3610- Design d-1 636
1034 1403 1772 2141 2510 2879 3248 3617 20 mRNA 222-230 637- 1035-
1404- 1773- 2142- 2511- 2880- 3249- 3618- Design d-2 645 1042 1411
1780 2149 2518 2887 3256 3625 21 mRNA 231-239 646- 1043- 1412-
1781- 2150- 2519- 2888- 3257- 3626- Design d-3 654 1050 1419 1788
2157 2526 2895 3264 3633 22 mRNA 240-248 655- 1051- 1420- 1789-
2158- 2527- 2896- 3265- 3634- Design d-4 663 1058 1427 1796 2165
2534 2903 3272 3641 23 mRNA 249-257 664- 1059- 1428- 1797- 2166-
2535- 2904- 3273- 3642- Design d-5 672 1066 1435 1804 2173 2542
2911 3280 3649 24 mRNA 258-266 673- 1067- 1436- 1805- 2174- 2543-
2912- 3281- 3650- Design e-1 681 1074 1443 1812 2181 2550 2919 3288
3657 25 mRNA 267-275 682- 1075- 1444- 1813- 2182- 2551- 2920- 3289-
3658- Design e-2 690 1082 1451 1820 2189 2558 2927 3296 3665 26
mRNA 276-284 691- 1083- 1452- 1821- 2190- 2559- 2928- 3297- 3666-
Design e-3 699 1090 1459 1828 2197 2566 2935 3304 3673 27 mRNA
285-293 700- 1091- 1460- 1829- 2198- 2567- 2936- 3305- 3674- Design
e-4 708 1098 1467 1836 2205 2574 2943 3312 3681 28 mRNA 294-302
709- 1099- 1468- 1837- 2206- 2575- 2944- 3313- 3682- Design e-5 717
1106 1475 1844 2213 2582 2951 3320 3689 29 mRNA 303-311 718- 1107-
1476- 1845- 2214- 2583- 2952- 3321- 3690- Design e-6 726 1114 1483
1852 2221 2590 2959 3328 3697 30 mRNA 312-320 727- 1115- 1484-
1853- 2222- 2591- 2960- 3329- 3698- Design f-1 735 1122 1491 1860
2229 2598 2967 3336 3705 31 mRNA 321-329 736- 1123- 1492- 1861-
2230- 2599- 2968- 3337- 3706- Design f-2 744 1130 1499 1868 2237
2606 2975 3344 3713 32 mRNA 330-338 745- 1131- 1500- 1869- 2238-
2607- 2976- 3345- 3714- Design f-3 753 1138 1507 1876 2245 2614
2983 3352 3721 33 mRNA 339-347 754- 1139- 1508- 1877- 2246- 2615-
2984- 3353- 3722- Design f-4 762 1146 1515 1884 2253 2622 2991 3360
3729 34 mRNA 348-356 763- 1147- 1516- 1885- 2254- 2623- 2992- 3361-
3730- Design f-5 771 1154 1523 1892 2261 2630 2999 3368 3737 35
mRNA 357-365 772- 1155- 1524- 1893- 2262- 2631- 3000- 3369- 3738-
Design g-1 780 1162 1531 1900 2269 2638 3007 3376 3745 36 mRNA
366-374 781- 1163- 1532- 1901- 2270- 2639- 3008- 3377- 3746- Design
g-2 789 1170 1539 1908 2277 2646 3015 3384 3753 37 mRNA 375-383
790- 1171- 1540- 1909- 2278- 2647- 3016- 3385- 3754- Design g-3 798
1178 1547 1916 2285 2654 3023 3392 3761 38 mRNA 384-392 799- 1179-
1548- 1917- 2286- 2655- 3024- 3393- 3762- Design g-4 807 1186 1555
1924 2293 2662 3031 3400 3769 39 mRNA 393-401 808- 1187- 1556-
1925- 2294- 2663- 3032- 3401- 3770- Design g-5 816 1194 1563 1932
2301 2670 3039 3408 3777 40 mRNA 402-410 817- 1195- 1564- 1933-
2302- 2671- 3040- 3409- 3778- Design h-1 825 1202 1571 1940 2309
2678 3047 3416 3785 41 mRNA 411-419 826- 1203- 1572- 1941- 2310-
2679- 3048- 3417- 3786- Design h-2 834 1210 1579 1948 2317 2686
3055 3424 3793 42 mRNA 420-428 835- 1211- 1580- 1949- 2318- 2687-
3056- 3425- 3794- Design h-3 843 1218 1587 1956 2325 2694 3063 3432
3801 43 mRNA 429-437 844- 1219- 1588- 1957- 2326- 2695- 3064- 3433-
3802- Design h-4 852 1226 1595 1964 2333 2702 3071 3440 3809 44
mRNA 438-446 853- 1227- 1596- 1965- 2334- 2703- 3072- 3441- 3810-
Design h-5 861 1234 1603 1972 2341 2710 3079 3448 3817 45 mRNA
447-455 862- 1235- 1604- 1973- 2342- 2711- 3080- 3449- 3818- Design
i-1 870 1242 1611 1980 2349 2718 3087 3456 3825 46 mRNA 456-473
871- 1243- 1612- 1981- 2350- 2719- 3088- 3457- 3826- Design i-2 888
1258 1627 1996 2365 2734 3103 3472 3841 47 mRNA 474-482 889- 1259-
1628- 1997- 2366- 2735- 3104- 3473- 3842- Design i-3 897 1266 1635
2004 2373 2742 3111 3480 3849 48 mRNA 8278 Design i-4
TABLE-US-00008 TABLE 5B Preferred mRNA constructs encoding RSV F
(columns A-J), derived from HRSV(Memphis-37) 1 A B C D E F G H I J
2 Protein 11726 12095 12464 12833 13940 14309 14678 15047 15416
15785 3 cds 11727- 12096- 12465- 12834- 13941- 14310- 14679- 15048-
15417- 15786- 11734, 12103, 12472, 12841, 13948, 14317, 14686,
15055, 15424, 15793, 21389 21390 21391 21392 21395 21396 21397
21398 21399 21400 4 mRNA 11735- 12104- 12473- 12842- 13949- 14318-
14687- 15056- 15425- 15794- Design a-1 11742, 12111, 12480, 12849,
13956, 14325, 14694, 15063, 15432, 15801, 21489, 21491, 21493-
21496- 21505- 21508- 21511- 21514- 21517- 21520- 21490, 21492,
21495, 21498, 21507, 21510, 21513, 21516, 21519, 21522, 21635,
21637, 21639- 21642- 21651- 21654- 21657- 21660- 21663- 21666-
21636 21638 21641 21644 21653 21656 21659 21662 21665 21668 5 mRNA
11743- 12112- 12481- 12850- 13957- 14326- 14695- 15064- 15433-
15802- Design a-2 11750 12119 12488 12857 13964 14333 14702 15071
15440 15809 6 mRNA 11751- 12120- 12489- 12858- 13965- 14334- 14703-
15072- 15441- 15810- Design a-3 11758 12127 12496 12865 13972 14341
14710 15079 15448 15817 7 mRNA 11759- 12128- 12497- 12866- 13973-
14342- 14711- 15080- 15449- 15818- Design a-4 11766 12135 12504
12873 13980 14349 14718 15087 15456 15825 8 mRNA 11767- 12136-
12505- 12874- 13981- 14350- 14719- 15088- 15457- 15826- Design a-5
11774 12143 12512 12881 13988 14357 14726 15095 15464 15833 9 mRNA
11775- 12144- 12513- 12882- 13989- 14358- 14727- 15096- 15465-
15834- Design b-1 11782 12151 12520 12889 13996 14365 14734 15103
15472 15841 10 mRNA 11783- 12152- 12521- 12890- 13997- 14366-
14735- 15104- 15473- 15842- Design b-2 11790 12159 12528 12897
14004 14373 14742 15111 15480 15849 11 mRNA 11791- 12160- 12529-
12898- 14005- 14374- 14743- 15112- 15481- 15850- Design b-3 11798
12167 12536 12905 14012 14381 14750 15119 15488 15857 12 mRNA
11799- 12168- 12537- 12906- 14013- 14382- 14751- 15120- 15489-
15858- Design b-4 11806 12175 12544 12913 14020 14389 14758 15127
15496 15865 13 mRNA 11807- 12176- 12545- 12914- 14021- 14390-
14759- 15128- 15497- 15866- Design b-5 11814 12183 12552 12921
14028 14397 14766 15135 15504 15873 14 mRNA 11815- 12184- 12553-
12922- 14029- 14398- 14767- 15136- 15505- 15874- Design c-1 11822
12191 12560 12929 14036 14405 14774 15143 15512 15881 15 mRNA
11823- 12192- 12561- 12930- 14037- 14406- 14775- 15144- 15513-
15882- Design c-2 11830 12199 12568 12937 14044 14413 14782 15151
15520 15889 16 mRNA 11831- 12200- 12569- 12938- 14045- 14414-
14783- 15152- 15521- 15890- Design c-3 11838 12207 12576 12945
14052 14421 14790 15159 15528 15897 17 mRNA 11839- 12208- 12577-
12946- 14053- 14422- 14791- 15160- 15529- 15898- Design c-4 11846
12215 12584 12953 14060 14429 14798 15167 15536 15905 18 mRNA
11847- 12216- 12585- 12954- 14061- 14430- 14799- 15168- 15537-
15906- Design c-5 11854 12223 12592 12961 14068 14437 14806 15175
15544 15913 19 mRNA 11855- 12224- 12593- 12962- 14069- 14438-
14807- 15176- 15545- 15914- Design d-1 11862 12231 12600 12969
14076 14445 14814 15183 15552 15921 20 mRNA 11863- 12232- 12601-
12970- 14077- 14446- 14815- 15184- 15553- 15922- Design d-2 11870
12239 12608 12977 14084 14453 14822 15191 15560 15929 21 mRNA
11871- 12240- 12609- 12978- 14085- 14454- 14823- 15192- 15561-
15930- Design d-3 11878 12247 12616 12985 14092 14461 14830 15199
15568 15937 22 mRNA 11879- 12248- 12617- 12986- 14093- 14462-
14831- 15200- 15569- 15938- Design d-4 11886 12255 12624 12993
14100 14469 14838 15207 15576 15945 23 mRNA 11887- 12256- 12625-
12994- 14101- 14470- 14839- 15208- 15577- 15946- Design d-5 11894
12263 12632 13001 14108 14477 14846 15215 15584 15953 24 mRNA
11895- 12264- 12633- 13002- 14109- 14478- 14847- 15216- 15585-
15954- Design e-1 11902 12271 12640 13009 14116 14485 14854 15223
15592 15961 25 mRNA 11903- 12272- 12641- 13010- 14117- 14486-
14855- 15224- 15593- 15962- Design e-2 11910 12279 12648 13017
14124 14493 14862 15231 15600 15969 26 mRNA 11911- 12280- 12649-
13018- 14125- 14494- 14863- 15232- 15601- 15970- Design e-3 11918
12287 12656 13025 14132 14501 14870 15239 15608 15977 27 mRNA
11919- 12288- 12657- 13026- 14133- 14502- 14871- 15240- 15609-
15978- Design e-4 11926 12295 12664 13033 14140 14509 14878 15247
15616 15985 28 mRNA 11927- 12296- 12665- 13034- 14141- 14510-
14879- 15248- 15617- 15986- Design e-5 11934 12303 12672 13041
14148 14517 14886 15255 15624 15993 29 mRNA 11935- 12304- 12673-
13042- 14149- 14518- 14887- 15256- 15625- 15994- Design e-6 11942
12311 12680 13049 14156 14525 14894 15263 15632 16001 30 mRNA
11943- 12312- 12681- 13050- 14157- 14526- 14895- 15264- 15633-
16002- Design f-1 11950 12319 12688 13057 14164 14533 14902 15271
15640 16009 31 mRNA 11951- 12320- 12689- 13058- 14165- 14534-
14903- 15272- 15641- 16010- Design f-2 11958 12327 12696 13065
14172 14541 14910 15279 15648 16017 32 mRNA 11959- 12328- 12697-
13066- 14173- 14542- 14911- 15280- 15649- 16018- Design f-3 11966
12335 12704 13073 14180 14549 14918 15287 15656 16025 33 mRNA
11967- 12336- 12705- 13074- 14181- 14550- 14919- 15288- 15657-
16026- Design f-4 11974 12343 12712 13081 14188 14557 14926 15295
15664 16033 34 mRNA 11975- 12344- 12713- 13082- 14189- 14558-
14927- 15296- 15665- 16034- Design f-5 11982 12351 12720 13089
14196 14565 14934 15303 15672 16041 35 mRNA 11983- 12352- 12721-
13090- 14197- 14566- 14935- 15304- 15673- 16042- Design g-1 11990
12359 12728 13097 14204 14573 14942 15311 15680 16049 36 mRNA
11991- 12360- 12729- 13098- 14205- 14574- 14943- 15312- 15681-
16050- Design g-2 11998 12367 12736 13105 14212 14581 14950 15319
15688 16057 37 mRNA 11999- 12368- 12737- 13106- 14213- 14582-
14951- 15320- 15689- 16058- Design g-3 12006 12375 12744 13113
14220 14589 14958 15327 15696 16065 38 mRNA 12007- 12376- 12745-
13114- 14221- 14590- 14959- 15328- 15697- 16066- Design g-4 12014
12383 12752 13121 14228 14597 14966 15335 15704 16073 39 mRNA
12015- 12384- 12753- 13122- 14229- 14598- 14967- 15336- 15705-
16074- Design g-5 12022 12391 12760 13129 14236 14605 14974 15343
15712 16081 40 mRNA 12023- 12392- 12761- 13130- 14237- 14606-
14975- 15344- 15713- 16082- Design h-1 12030 12399 12768 13137
14244 14613 14982 15351 15720 16089 41 mRNA 12031- 12400- 12769-
13138- 14245- 14614- 14983- 15352- 15721- 16090- Design h-2 12038
12407 12776 13145 14252 14621 14990 15359 15728 16097 42 mRNA
12039- 12408- 12777- 13146- 14253- 14622- 14991- 15360- 15729-
16098- Design h-3 12046 12415 12784 13153 14260 14629 14998 15367
15736 16105 43 mRNA 12047- 12416- 12785- 13154- 14261- 14630-
14999- 15368- 15737- 16106- Design h-4 12054 12423 12792 13161
14268 14637 15006 15375 15744 16113 44 mRNA 12055- 12424- 12793-
13162- 14269- 14638- 15007- 15376- 15745- 16114- Design h-5 12062
12431 12800 13169 14276 14645 15014 15383 15752 16121 45 mRNA
12063- 12432- 12801- 13170- 14277- 14646- 15015- 15384- 15753-
16122- Design i-1 12070 12439 12808 13177 14284 14653 15022 15391
15760 16129 46 mRNA 12071- 12440- 12809- 13178- 14285- 14654-
15023- 15392- 15761- 16130- Design i-2 12086 12455 12824 13193
14300 14669 15038 15407 15776 16145 47 mRNA 12087- 12456- 12825-
13194- 14301- 14670- 15039- 15408- 15777- 16146- Design i-3 12094
12463 12832 13201 14308 14677 15046 15415 15784 16153
[0402] In Table 6A and 6B, the protein designs are indicated in row
1. Therein, Columns K to V represent specific suitable constructs
of the invention derived from RSV Fusion (F) protein, wherein
Column K provides suitable sequences for F_DSCav1_mut0, Column L
provides suitable sequences for F-del_DSCav1_mut0, Column M
provides suitable sequences for F_DSCav1_mut4, Column N provides
suitable sequences for F-del_DSCav1_mut4, Column O provides
suitable sequences for F_DSCav1_mut5, Column P provides suitable
sequences for F-del_DSCav1_mut5, Column Q provides suitable
sequences for F_DSCav1_mut6, Column R provides suitable sequences
for F-del_DSCav1_mut6, Column S provides suitable sequences for
F_DSCav1_mut7, Column T provides suitable sequences for
F-del_DSCav1_mut7, Column U provides suitable sequences for
F_DSCav1_mut8, Column V provides suitable sequences for
F-del_DSCav1_mut8.
[0403] The protein designs are indicated in the first row (Columns
K to V), the specific protein SEQ ID NOs as provided in the
sequence listing are in row 2, "PRT". The SEQ ID NOs of
corresponding coding sequences for each protein construct are
provided in row 3, "cds" (compare with Table 4 for different cds
optimizations). Further information e.g. regarding the type of
codon modified coding sequence (opt1, opt2, opt3, opt4, opt5, opt6,
opt11 etc.) is provided in the <223> identifier of the
respective SEQ ID NO in the sequence listing and in Table 4. The
SEQ ID NOs of corresponding mRNA constructs comprising said coding
sequences and suitable 3'-UTRs and 5'-UTRs according to the
invention are provided in each following row (mRNA designs "a-1" to
"i-3" as specified herein).
[0404] In Table 6A, mRNA sequences derived from HRSV(A2) are
provided, in Table 6B mRNA sequences derived from HRSV(Memphis-37)
are provided
[0405] Further information e.g. regarding the type of coding
sequence (wt, opt1, opt2, opt3, opt4, opt5, opt6, opt11 etc.)
comprised in the mRNA constructs is provided in the <223>
identifier of the respective SEQ ID NO in the sequence listing.
TABLE-US-00009 TABLE 6A Preferred mRNA constructs encoding RSV F
(column K-V), derived from HRSV(A2) K L M N O P Q R S T U V PRT
3850 4219 4588 4957 5326 5695 6064 6433 6802 7171 7540 7909 cds
3851- 4220- 4589- 4958- 5327- 5696- 6065- 6434- 6803- 7172- 7541-
7910- 3858, 4227, 4596, 4965, 5334, 5703, 6072, 6441, 6810, 7179,
7548, 7917, 21367 21368 21375 21376 21377 21378 21379 21380 21381
21382 21383 21384 a-1 3859- 4228- 4597- 4966- 5335- 5704- 6073-
6442- 6811- 7180- 7549- 7918- 3866, 4235, 4604, 4973, 5342, 5711,
6080, 6449, 6818, 7187, 7556, 7925, 21427- 21430- 21451- 21454-
21457- 21460- 21463- 21466- 21469- 21472- 21475- 21478- 21429,
21432, 21453, 21456, 21459, 21462, 21465, 21468, 21471, 21474,
21477, 21480, 21573- 21576- 21597- 21600- 21603- 21606- 21609-
21612- 21615- 21618- 21621- 21624- 21575 21578 21599 21602 21605
21608 21611 21614 21617 21620 21623 21626 a-2 3867- 4236- 4605-
4974- 5343- 5712- 6081- 6450- 6819- 7188- 7557- 7926- 3874 4243
4612 4981 5350 5719 6088 6457 6826 7195 7564 7933 a-3 3875- 4244-
4613- 4982- 5351- 5720- 6089- 6458- 6827- 7196- 7565- 7934- 3882
4251 4620 4989 5358 5727 6096 6465 6834 7203 7572 7941 a-4 3883-
4252- 4621- 4990- 5359- 5728- 6097- 6466- 6835- 7204- 7573- 7942-
3890 4259 4628 4997 5366 5735 6104 6473 6842 7211 7580 7949 a-5
3891- 4260- 4629- 4998- 5367- 5736- 6105- 6474- 6843- 7212- 7581-
7950- 3898 4267 4636 5005 5374 5743 6112 6481 6850 7219 7588 7957
b-1 3899- 4268- 4637- 5006- 5375- 5744- 6113- 6482- 6851- 7220-
7589- 7958- 3906 4275 4644 5013 5382 5751 6120 6489 6858 7227 7596
7965 b-2 3907- 4276- 4645- 5014- 5383- 5752- 6121- 6490- 6859-
7228- 7597- 7966- 3914 4283 4652 5021 5390 5759 6128 6497 6866 7235
7604 7973 b-3 3915- 4284- 4653- 5022- 5391- 5760- 6129- 6498- 6867-
7236- 7605- 7974- 3922 4291 4660 5029 5398 5767 6136 6505 6874 7243
7612 7981 b-4 3923- 4292- 4661- 5030- 5399- 5768- 6137- 6506- 6875-
7244- 7613- 7982- 3930 4299 4668 5037 5406 5775 6144 6513 6882 7251
7620 7989 b-5 3931- 4300- 4669- 5038- 5407- 5776- 6145- 6514- 6883-
7252- 7621- 7990- 3938 4307 4676 5045 5414 5783 6152 6521 6890 7259
7628 7997 c-1 3939- 4308- 4677- 5046- 5415- 5784- 6153- 6522- 6891-
7260- 7629- 7998- 3946 4315 4684 5053 5422 5791 6160 6529 6898 7267
7636 8005 c-2 3947- 4316- 4685- 5054- 5423- 5792- 6161- 6530- 6899-
7268- 7637- 8006- 3954 4323 4692 5061 5430 5799 6168 6537 6906 7275
7644 8013 c-3 3955- 4324- 4693- 5062- 5431- 5800- 6169- 6538- 6907-
7276- 7645- 8014- 3962 4331 4700 5069 5438 5807 6176 6545 6914 7283
7652 8021 c-4 3963- 4332- 4701- 5070- 5439- 5808- 6177- 6546- 6915-
7284- 7653- 8022- 3970 4339 4708 5077 5446 5815 6184 6553 6922 7291
7660 8029 c-5 3971- 4340- 4709- 5078- 5447- 5816- 6185- 6554- 6923-
7292- 7661- 8030- 3978 4347 4716 5085 5454 5823 6192 6561 6930 7299
7668 8037 d-1 3979- 4348- 4717- 5086- 5455- 5824- 6193- 6562- 6931-
7300- 7669- 8038- 3986 4355 4724 5093 5462 5831 6200 6569 6938 7307
7676 8045 d-2 3987- 4356- 4725- 5094- 5463- 5832- 6201- 6570- 6939-
7308- 7677- 8046- 3994 4363 4732 5101 5470 5839 6208 6577 6946 7315
7684 8053 d-3 3995- 4364- 4733- 5102- 5471- 5840- 6209- 6578- 6947-
7316- 7685- 8054- 4002 4371 4740 5109 5478 5847 6216 6585 6954 7323
7692 8061 d-4 4003- 4372- 4741- 5110- 5479- 5848- 6217- 6586- 6955-
7324- 7693- 8062- 4010 4379 4748 5117 5486 5855 6224 6593 6962 7331
7700 8069 d-5 4011- 4380- 4749- 5118- 5487- 5856- 6225- 6594- 6963-
7332- 7701- 8070- 4018 4387 4756 5125 5494 5863 6232 6601 6970 7339
7708 8077 e-1 4019- 4388- 4757- 5126- 5495- 5864- 6233- 6602- 6971-
7340- 7709- 8078- 4026 4395 4764 5133 5502 5871 6240 6609 6978 7347
7716 8085 e-2 4027- 4396- 4765- 5134- 5503- 5872- 6241- 6610- 6979-
7348- 7717- 8086- 4034 4403 4772 5141 5510 5879 6248 6617 6986 7355
7724 8093 e-3 4035- 4404- 4773- 5142- 5511- 5880- 6249- 6618- 6987-
7356- 7725- 8094- 4042 4411 4780 5149 5518 5887 6256 6625 6994 7363
7732 8101 e-4 4043- 4412- 4781- 5150- 5519- 5888- 6257- 6626- 6995-
7364- 7733- 8102- 4050 4419 4788 5157 5526 5895 6264 6633 7002 7371
7740 8109 e-5 4051- 4420- 4789- 5158- 5527- 5896- 6265- 6634- 7003-
7372- 7741- 8110- 4058 4427 4796 5165 5534 5903 6272 6641 7010 7379
7748 8117 e-6 4059- 4428- 4797- 5166- 5535- 5904- 6273- 6642- 7011-
7380- 7749- 8118- 4066 4435 4804 5173 5542 5911 6280 6649 7018 7387
7756 8125 f-1 4067- 4436- 4805- 5174- 5543- 5912- 6281- 6650- 7019-
7388- 7757- 8126- 4074 4443 4812 5181 5550 5919 6288 6657 7026 7395
7764 8133 f-2 4075- 4444- 4813- 5182- 5551- 5920- 6289- 6658- 7027-
7396- 7765- 8134- 4082 4451 4820 5189 5558 5927 6296 6665 7034 7403
7772 8141 f-3 4083- 4452- 4821- 5190- 5559- 5928- 6297- 6666- 7035-
7404- 7773- 8142- 4090 4459 4828 5197 5566 5935 6304 6673 7042 7411
7780 8149 f-4 4091- 4460- 4829- 5198- 5567- 5936- 6305- 6674- 7043-
7412- 7781- 8150- 4098 4467 4836 5205 5574 5943 6312 6681 7050 7419
7788 8157 f-5 4099- 4468- 4837- 5206- 5575- 5944- 6313- 6682- 7051-
7420- 7789- 8158- 4106 4475 4844 5213 5582 5951 6320 6689 7058 7427
7796 8165 g-1 4107- 4476- 4845- 5214- 5583- 5952- 6321- 6690- 7059-
7428- 7797- 8166- 4114 4483 4852 5221 5590 5959 6328 6697 7066 7435
7804 8173 g-2 4115- 4484- 4853- 5222- 5591- 5960- 6329- 6698- 7067-
7436- 7805- 8174- 4122 4491 4860 5229 5598 5967 6336 6705 7074 7443
7812 8181 g-3 4123- 4492- 4861- 5230- 5599- 5968- 6337- 6706- 7075-
7444- 7813- 8182- 4130 4499 4868 5237 5606 5975 6344 6713 7082 7451
7820 8189 g-4 4131- 4500- 4869- 5238- 5607- 5976- 6345- 6714- 7083-
7452- 7821- 8190- 4138 4507 4876 5245 5614 5983 6352 6721 7090 7459
7828 8197 g-5 4139- 4508- 4877- 5246- 5615- 5984- 6353- 6722- 7091-
7460- 7829- 8198- 4146 4515 4884 5253 5622 5991 6360 6729 7098 7467
7836 8205 h-1 4147- 4516- 4885- 5254- 5623- 5992- 6361- 6730- 7099-
7468- 7837- 8206- 4154 4523 4892 5261 5630 5999 6368 6737 7106 7475
7844 8213 h-2 4155- 4524- 4893- 5262- 5631- 6000- 6369- 6738- 7107-
7476- 7845- 8214- 4162 4531 4900 5269 5638 6007 6376 6745 7114 7483
7852 8221 h-3 4163- 4532- 4901- 5270- 5639- 6008- 6377- 6746- 7115-
7484- 7853- 8222- 4170 4539 4908 5277 5646 6015 6384 6753 7122 7491
7860 8229 h-4 4171- 4540- 4909- 5278- 5647- 6016- 6385- 6754- 7123-
7492- 7861- 8230- 4178 4547 4916 5285 5654 6023 6392 6761 7130 7499
7868 8237 h-5 4179- 4548- 4917- 5286- 5655- 6024- 6393- 6762- 7131-
7500- 7869- 8238- 4186 4555 4924 5293 5662 6031 6400 6769 7138 7507
7876 8245 i-1 4187- 4556- 4925- 5294- 5663- 6032- 6401- 6770- 7139-
7508- 7877- 8246- 4194 4563 4932 5301 5670 6039 6408 6777 7146 7515
7884 8253 i-2 4195- 4564- 4933- 5302- 5671- 6040- 6409- 6778- 7147-
7516- 7885- 8254- 4210 4579 4948 5317 5686 6055 6424 6793 7162 7531
7900 8269 i-3 4211- 4580- 4949- 5318- 5687- 6056- 6425- 6794- 7163-
7532- 7901- 8270- 4218 4587 4956 5325 5694 6063 6432 6801 7170 7539
7908 8277
TABLE-US-00010 TABLE 6B Preferred mRNA constructs encoding RSV F
(column K-V), derived from HRSV(Memphis-37) K L M N O P Q R S T U V
PRT 13202 13571 16154 16523 16892 17261 17630 17999 18368 18737
19106 19475 cds 13203- 13572- 16155- 16524- 16893- 17262- 17631-
18000- 18369- 18738- 19107- 19476- 13210, 13579, 16162, 16531,
16900, 17269, 17638, 18007, 18376, 18745, 19114, 19483, 21393 21394
21401 21402 21403 21404 21405 21406 21407 21408 21409 21410 a-1
13211- 13580- 16163- 16532- 16901- 17270- 17639- 18008- 18377-
18746- 19115- 19484- 13218, 13587, 16170, 16539, 16908, 17277,
17646, 18015, 18384, 18753, 19122, 19491, 21499- 21502- 21523-
21526- 21529- 21532- 21535- 21538- 21541- 21544- 21547- 21550-
21501, 21504, 21525, 21528, 21531, 21534, 21537, 21540, 21543,
21546, 21549, 21552, 21645- 21648- 21669- 21672- 21675- 21678-
21681- 21684- 21687- 21690- 21693- 21696- 21647 21650 21671 21674
21677 21680 21683 21686 21689 21692 21695 21698 a-2 13219- 13588-
16171- 16540- 16909- 17278- 17647- 18016- 18385- 18754- 19123-
19492- 13226 13595 16178 16547 16916 17285 17654 18023 18392 18761
19130 19499 a-3 13227- 13596- 16179- 16548- 16917- 17286- 17655-
18024- 18393- 18762- 19131- 19500- 13234 13603 16186 16555 16924
17293 17662 18031 18400 18769 19138 19507 a-4 13235- 13604- 16187-
16556- 16925- 17294- 17663- 18032- 18401- 18770- 19139- 19508-
13242 13611 16194 16563 16932 17301 17670 18039 18408 18777 19146
19515 a-5 13243- 13612- 16195- 16564- 16933- 17302- 17671- 18040-
18409- 18778- 19147- 19516- 13250 13619 16202 16571 16940 17309
17678 18047 18416 18785 19154 19523 b-1 13251- 13620- 16203- 16572-
16941- 17310- 17679- 18048- 18417- 18786- 19155- 19524- 13258 13627
16210 16579 16948 17317 17686 18055 18424 18793 19162 19531 b-2
13259- 13628- 16211- 16580- 16949- 17318- 17687- 18056- 18425-
18794- 19163- 19532- 13266 13635 16218 16587 16956 17325 17694
18063 18432 18801 19170 19539 b-3 13267- 13636- 16219- 16588-
16957- 17326- 17695- 18064- 18433- 18802- 19171- 19540- 13274 13643
16226 16595 16964 17333 17702 18071 18440 18809 19178 19547 b-4
13275- 13644- 16227- 16596- 16965- 17334- 17703- 18072- 18441-
18810- 19179- 19548- 13282 13651 16234 16603 16972 17341 17710
18079 18448 18817 19186 19555 b-5 13283- 13652- 16235- 16604-
16973- 17342- 17711- 18080- 18449- 18818- 19187- 19556- 13290 13659
16242 16611 16980 17349 17718 18087 18456 18825 19194 19563 c-1
13291- 13660- 16243- 16612- 16981- 17350- 17719- 18088- 18457-
18826- 19195- 19564- 13298 13667 16250 16619 16988 17357 17726
18095 18464 18833 19202 19571 c-2 13299- 13668- 16251- 16620-
16989- 17358- 17727- 18096- 18465- 18834- 19203- 19572- 13306 13675
16258 16627 16996 17365 17734 18103 18472 18841 19210 19579 c-3
13307- 13676- 16259- 16628- 16997- 17366- 17735- 18104- 18473-
18842- 19211- 19580- 13314 13683 16266 16635 17004 17373 17742
18111 18480 18849 19218 19587 c-4 13315- 13684- 16267- 16636-
17005- 17374- 17743- 18112- 18481- 18850- 19219- 19588- 13322 13691
16274 16643 17012 17381 17750 18119 18488 18857 19226 19595 c-5
13323- 13692- 16275- 16644- 17013- 17382- 17751- 18120- 18489-
18858- 19227- 19596- 13330 13699 16282 16651 17020 17389 17758
18127 18496 18865 19234 19603 d-1 13331- 13700- 16283- 16652-
17021- 17390- 17759- 18128- 18497- 18866- 19235- 19604- 13338 13707
16290 16659 17028 17397 17766 18135 18504 18873 19242 19611 d-2
13339- 13708- 16291- 16660- 17029- 17398- 17767- 18136- 18505-
18874- 19243- 19612- 13346 13715 16298 16667 17036 17405 17774
18143 18512 18881 19250 19619 d-3 13347- 13716- 16299- 16668-
17037- 17406- 17775- 18144- 18513- 18882- 19251- 19620- 13354 13723
16306 16675 17044 17413 17782 18151 18520 18889 19258 19627 d-4
13355- 13724- 16307- 16676- 17045- 17414- 17783- 18152- 18521-
18890- 19259- 19628- 13362 13731 16314 16683 17052 17421 17790
18159 18528 18897 19266 19635 d-5 13363- 13732- 16315- 16684-
17053- 17422- 17791- 18160- 18529- 18898- 19267- 19636- 13370 13739
16322 16691 17060 17429 17798 18167 18536 18905 19274 19643 e-1
13371- 13740- 16323- 16692- 17061- 17430- 17799- 18168- 18537-
18906- 19275- 19644- 13378 13747 16330 16699 17068 17437 17806
18175 18544 18913 19282 19651 e-2 13379- 13748- 16331- 16700-
17069- 17438- 17807- 18176- 18545- 18914- 19283- 19652- 13386 13755
16338 16707 17076 17445 17814 18183 18552 18921 19290 19659 e-3
13387- 13756- 16339- 16708- 17077- 17446- 17815- 18184- 18553-
18922- 19291- 19660- 13394 13763 16346 16715 17084 17453 17822
18191 18560 18929 19298 19667 e-4 13395- 13764- 16347- 16716-
17085- 17454- 17823- 18192- 18561- 18930- 19299- 19668- 13402 13771
16354 16723 17092 17461 17830 18199 18568 18937 19306 19675 e-5
13403- 13772- 16355- 16724- 17093- 17462- 17831- 18200- 18569-
18938- 19307- 19676- 13410 13779 16362 16731 17100 17469 17838
18207 18576 18945 19314 19683 e-6 13411- 13780- 16363- 16732-
17101- 17470- 17839- 18208- 18577- 18946- 19315- 19684- 13418 13787
16370 16739 17108 17477 17846 18215 18584 18953 19322 19691 f-1
13419- 13788- 16371- 16740- 17109- 17478- 17847- 18216- 18585-
18954- 19323- 19692- 13426 13795 16378 16747 17116 17485 17854
18223 18592 18961 19330 19699 f-2 13427- 13796- 16379- 16748-
17117- 17486- 17855- 18224- 18593- 18962- 19331- 19700- 13434 13803
16386 16755 17124 17493 17862 18231 18600 18969 19338 19707 f-3
13435- 13804- 16387- 16756- 17125- 17494- 17863- 18232- 18601-
18970- 19339- 19708- 13442 13811 16394 16763 17132 17501 17870
18239 18608 18977 19346 19715 f-4 13443- 13812- 16395- 16764-
17133- 17502- 17871- 18240- 18609- 18978- 19347- 19716- 13450 13819
16402 16771 17140 17509 17878 18247 18616 18985 19354 19723 f-5
13451- 13820- 16403- 16772- 17141- 17510- 17879- 18248- 18617-
18986- 19355- 19724- 13458 13827 16410 16779 17148 17517 17886
18255 18624 18993 19362 19731 g-1 13459- 13828- 16411- 16780-
17149- 17518- 17887- 18256- 18625- 18994- 19363- 19732- 13466 13835
16418 16787 17156 17525 17894 18263 18632 19001 19370 19739 g-2
13467- 13836- 16419- 16788- 17157- 17526- 17895- 18264- 18633-
19002- 19371- 19740- 13474 13843 16426 16795 17164 17533 17902
18271 18640 19009 19378 19747 g-3 13475- 13844- 16427- 16796-
17165- 17534- 17903- 18272- 18641- 19010- 19379- 19748- 13482 13851
16434 16803 17172 17541 17910 18279 18648 19017 19386 19755 g-4
13483- 13852- 16435- 16804- 17173- 17542- 17911- 18280- 18649-
19018- 19387- 19756- 13490 13859 16442 16811 17180 17549 17918
18287 18656 19025 19394 19763 g-5 13491- 13860- 16443- 16812-
17181- 17550- 17919- 18288- 18657- 19026- 19395- 19764- 13498 13867
16450 16819 17188 17557 17926 18295 18664 19033 19402 19771 h-1
13499- 13868- 16451- 16820- 17189- 17558- 17927- 18296- 18665-
19034- 19403- 19772- 13506 13875 16458 16827 17196 17565 17934
18303 18672 19041 19410 19779 h-2 13507- 13876- 16459- 16828-
17197- 17566- 17935- 18304- 18673- 19042- 19411- 19780- 13514 13883
16466 16835 17204 17573 17942 18311 18680 19049 19418 19787 h-3
13515- 13884- 16467- 16836- 17205- 17574- 17943- 18312- 18681-
19050- 19419- 19788- 13522 13891 16474 16843 17212 17581 17950
18319 18688 19057 19426 19795 h-4 13523- 13892- 16475- 16844-
17213- 17582- 17951- 18320- 18689- 19058- 19427- 19796- 13530 13899
16482 16851 17220 17589 17958 18327 18696 19065 19434 19803 h-5
13531- 13900- 16483- 16852- 17221- 17590- 17959- 18328- 18697-
19066- 19435- 19804- 13538 13907 16490 16859 17228 17597 17966
18335 18704 19073 19442 19811 i-1 13539- 13908- 16491- 16860-
17229- 17598- 17967- 18336- 18705- 19074- 19443- 19812- 13546 13915
16498 16867 17236 17605 17974 18343 18712 19081 19450 19819 i-2
13547- 13916- 16499- 16868- 17237- 17606- 17975- 18344- 18713-
19082- 19451- 19820- 13562 13931 16514 16883 17252 17621 17990
18359 18728 19097 19466 19835 i-3 13563- 13932- 16515- 16884-
17253- 17622- 17991- 18360- 18729- 19098- 19467- 19836- 13570 13939
16522 16891 17260 17629 17998 18367 18736 19105 19474 19843
[0406] In preferred embodiments, the artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of Seq ID NOs: 78-482, 493-897, 907-1266,
1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,
3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694,
5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908,
7918-8277, 8278, 21415-21480, 21561-21626, 11735-12094,
12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677,
14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570,
13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629,
17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474,
19484-19843, 21489-21552, 21635-21698 or a fragment or variant of
any of these sequences, wherein, optionally, at least one or more
than one, or wherein all uracil nucleotides are replaced by
pseudouridine (.psi.) nucleotides or N1-methylpseudouridine
(m1.psi.) nucleotides.
[0407] In preferred embodiments, the artificial RNA comprises (a)
at least one heterologous 5' untranslated region (5'-UTR) and/or at
least one heterologous 3' untranslated region (3'-UTR) and (b) at
least one coding sequence operably linked to said 3'-UTR and/or
5'-UTR encoding at least one antigenic peptide or protein derived
from a RSV F or a fragment or variant thereof, wherein [0408] said
artificial RNA comprises or consists of an RNA sequence which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
78-482, 11735-12094, 21415-21417, 21561-21563, 21489, 21490, 21635,
21636 (encoding F0) or a fragment or variant of any of these
sequences; [0409] said artificial RNA comprises or consists of an
RNA sequence which is identical or at least 70%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 493-897, 12104-12463, 21418-21420,
21564-21566, 21491, 21492, 21637, 21638 (encoding F-del) or a
fragment or variant of any of these sequences; [0410] said
artificial RNA comprises or consists of an RNA sequence which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
907-1266, 12473-12832, 21421-21423, 21567-21569, 21493-21495,
21639-21641 (encoding F0_DSCav1) or a fragment or variant of any of
these sequences; [0411] said artificial RNA comprises or consists
of an RNA sequence which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1276-1635, 8278, 12842-13201,
21424-21426, 21570-21572, 21496-21498, 21642-21644 (encoding
F-del_DSCav1) or a fragment or variant of any of these sequences;
[0412] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 1645-2004, 13949-14308, 21433-21435, 21579-21581, 21505-21507,
21651-21653 (encoding F_DSCav1_mut1) or a fragment or variant of
any of these sequences; [0413] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 2014-2373, 14318-14677,
21436-21438, 21582-21584, 21508-21510, 21654-21656 (encoding
F-del_DSCav1_mut1) or a fragment or variant of any of these
sequences; [0414] said artificial RNA comprises or consists of an
RNA sequence which is identical or at least 70%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 2383-2742, 14687-15046, 21439-21441,
21585-21587, 21511-21513, 21657-21659 (encoding F_DSCav1_mut2) or a
fragment or variant of any of these sequences; [0415] said
artificial RNA comprises or consists of an RNA sequence which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from the group consisting of SEQ ID NOs:
2752-3111, 15056-15415, 21442-21444, 21588-21590, 21514-21516,
21660-21662 (encoding F-del_DSCav1_mut2) or a fragment or variant
of any of these sequences; [0416] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 3121-3480, 15425-15784,
21445-21447, 21591-21593, 21517-21519, 21663-21665 (encoding
F_DSCav1_mut3) or a fragment or variant of any of these sequences;
[0417] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 3490-3849, 15794-16153, 21448-21450, 21594-21596, 21520-21522,
21666-21668 (encoding F-del_DSCav1_mut3) or a fragment or variant
of any of these sequences. [0418] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 3859-4218, 13211-13570,
21427-21429, 21573-21575, 21499-21501, 21645-21647 (encoding
F_DSCav1_mut0) or a fragment or variant of any of these sequences;
[0419] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 4228-4587, 13580-13939, 21430-21432, 21576-21578, 21502-21504,
21648-21650 (encoding F-del_DSCav1_mut0) or a fragment or variant
of any of these sequences; [0420] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 4597-4956, 16163-16522,
21451-21453, 21597-21599, 21523-21525, 21669-21671 (encoding
F_DSCav1_mut4) or a fragment or variant of any of these sequences;
[0421] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 4966-5325, 16532-16891, 21454-21456, 21600-21602, 21526-21528,
21672-21674 (encoding F-del_DSCav1_mut4) or a fragment or variant
of any of these sequences; [0422] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 5335-5694, 16901-17260,
21457-21459, 21603-21605, 21529-21531, 21675-21677 (encoding
F_DSCav1_mut5) or a fragment or variant of any of these sequences;
[0423] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 5704-6063, 17270-17629, 21460-21462, 21606-21608, 21532-21534,
21678-21680 (encoding F-del_DSCav1_mut5) or a fragment or variant
of any of these sequences; [0424] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 6073-6432, 17639-17998,
21463-21465, 21609-21611, 21535-21537, 21681-21683 (encoding
F_DSCav1_mut6) or a fragment or variant of any of these sequences;
[0425] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 6442-6801, 18008-18367, 21466-21468, 21612-21614, 21538-21540,
21684-21686 (encoding F-del_DSCav1_mut6) or a fragment or variant
of any of these sequences; [0426] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 6811-7170, 18377-18736,
21469-21471, 21615-21617, 21541-21543, 21687-21689 (encoding
F_DSCav1_mut7) or a fragment or variant of any of these sequences;
[0427] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 7180-7539, 18746-19105, 21472-21474, 21618-21620, 21544-21546,
21690-21692 (encoding F-del_DSCav1_mut7) or a fragment or variant
of any of these sequences; [0428] said artificial RNA comprises or
consists of an RNA sequence which is identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 7549-7908, 19115-19474,
21475-21477, 21621-21623, 21547-21549, 21693-21695 (encoding
F_DSCav1_mut8) or a fragment or variant of any of these sequences;
[0429] said artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 7918-8277, 19484-19843, 21478-21480, 21624-21626, 21550-21552,
21696-21698 (encoding F-del_DSCav1_mut8) or a fragment or variant
of any of these sequences, and wherein, optionally, at least one or
more than one, or wherein all uracil nucleotides are replaced by
pseudouridine (.psi.) nucleotides or N1-methylpseudouridine
(m1.psi.) nucleotides.
[0430] In particularly preferred embodiments, the artificial RNA
comprises (a) at least one heterologous 5' untranslated region
(5'-UTR) and/or at least one heterologous 3' untranslated region
(3'-UTR) and (b) at least one coding sequence operably linked to
said 3'-UTR and/or 5'-UTR encoding at least one antigenic peptide
or protein derived from a RSV F or a fragment or variant thereof,
wherein said artificial RNA comprises or consists of an RNA
sequence which is identical or at least 70%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 5704-6063, 17270-17629, 21460-21462,
21606-21608, 21532-21534, 21678-21680 (encoding F-del_DSCav1_mut5)
or a fragment or variant of any of these sequences.
[0431] As outlined throughout the specification, additional
information regarding suitable amino acid sequences or nucleic acid
sequences (coding sequences, mRNA sequences) may also be derived
from the sequence listing, in particular from the details provided
therein under identifier <223> as explained in the
following.
[0432] For example, the numeric identifier <223> in the
sequence listing of SEQ ID NO: 68 reads as follows: "derived and/or
modified protein sequence (artificial) from HRSV(A2)_F0". It has to
be noted that throughout the sequence listing, information provided
under numeric identifier <223> follows the same structure:
"<SEQUENCE_DESCRIPTOR> from <CONSTRUCT_IDENTIFIER>".
The <SEQUENCE_DESCRIPTOR> relates to the type of sequence
(e.g., "derived and/or modified protein sequence", "derived and/or
modified CDS" "mRNA product Design a-1 comprising derived and/or
modified sequence", or "mRNA product Design b-4 comprising derived
and/or modified sequence", or "mRNA product Design c-5 comprising
derived and/or modified sequence", or "mRNA product Design g-4
comprising derived and/or modified sequence" etc.) and whether the
sequence comprises or consists of a wild type sequence ("wt") or
whether the sequence comprises or consists of a sequence-optimized
sequence (e.g. "opt1", "opt2", "opt3", "opt4", "opt5", "opt6",
"opt11"; sequence optimizations are described in further detail
below). For example, the <SEQUENCE_DESCRIPTOR> provided under
numeric identifier <223> of SEQ ID NO: 68 reads as follows:
"derived and/or modified protein sequence (artificial)". The
<CONSTRUCT_IDENTIFIER> provided under numeric identifier
<223> has the following structures: ("organism_construct
name", or "organism__accession number__construct name") and is
intended to help the person skilled in the art to explicitly derive
suitable nucleic acid sequences (e.g., RNA, mRNA) encoding the same
RSV protein according to the invention. For example, the
<CONSTRUCT_IDENTIFIER> provided under numeric identifier
<223> of SEQ ID NO: 68 reads as follows: "HRSV(A2)_F0". In
that example, the respective protein sequence is derived from
"HRSV(A2)" (organism), wherein the protein comprises the structural
elements "F0" (construct name, full-length F). If the skilled
person uses the construct identifier of SEQ ID NO: 68, namely
"HRSV(A2)_F0", said person easily arrives at a list of suitable
nucleic acid coding sequences, e.g. RNA coding sequences and mRNA
sequences that can easily be retrieve from the sequence listing of
the present invention.
[0433] Composition:
[0434] A second aspect relates to a composition comprising at least
one artificial RNA of the first aspect.
[0435] In a preferred embodiment of the second aspect, the
composition comprises at least one artificial RNA of the first
aspect and, optionally, at least one pharmaceutically acceptable
carrier.
[0436] The term "pharmaceutically acceptable carrier" as used
herein preferably includes the liquid or non-liquid basis of the
composition. If the composition is provided in liquid form, the
carrier will preferably be water, typically pyrogen-free water;
isotonic saline or buffered (aqueous) solutions, e.g. phosphate,
citrate etc. buffered solutions. Water or preferably a buffer, more
preferably an aqueous buffer, may be used, containing a sodium
salt, preferably at least 50 mM of a sodium salt, a calcium salt,
preferably at least 0.01 mM of a calcium salt, and optionally a
potassium salt, preferably at least 3 mM of a potassium salt.
According to a preferred embodiment, the sodium, calcium and,
optionally, potassium salts may occur in the form of their
halogenides, e.g. chlorides, iodides, or bromides, in the form of
their hydroxides, carbonates, hydrogen carbonates, or sulfates,
etc. Without being limited thereto, examples of sodium salts
include e.g. NaCl, NaI, NaBr, Na.sub.2CO.sub.3, NaHC.sub.3,
Na.sub.2SO.sub.4, examples of the optional potassium salts include
e.g. KCl, KI, KBr, K.sub.2CO.sub.3, KHCO.sub.3, K.sub.2SO.sub.4,
and examples of calcium salts include e.g. CaCl.sub.2, CaI.sub.2,
CaBr.sub.2, CaCO.sub.3, CaSO.sub.4, Ca(OH).sub.2. Furthermore,
organic anions of the aforementioned cations may be contained in
the buffer.
[0437] In embodiments, the composition as defined herein may
comprise a plurality or at least more than one of the artificial
RNAs as defined in the context of the first aspect or the second
aspect of the invention.
[0438] In embodiments, the at least one RNA comprised in the
composition is a bi- or multicistronic nucleic acid, particularly a
bi- or multicistronic nucleic acid as defined herein, which encodes
the at least two, three, four, five, six, seven, eight, nine, ten,
eleven or twelve distinct antigenic peptides or protein derived
from the same RSV and/or a different RSV.
[0439] In embodiment, the composition may comprise at least 2, 3,
4, 5, 6, 7, 8, 9, 10 or even more different artificial RNAs as
defined in the context of the first aspect of the invention each
encoding at least one antigenic peptide or protein derived from
genetically the same RSV or a fragment or variant thereof. The
terms "same" or "same RSV" as used in the context of a virus, e.g.
"same virus", have to be understood as genetically the same.
Particularly, said (genetically) same virus expresses the same
proteins or peptides, wherein all proteins or peptides have the
same amino acid sequence. Particularly, said (genetically) same RSV
expresses essentially the same proteins, peptides or polyproteins,
wherein these protein, peptide or polyproteins preferably do not
differ in their amino acid sequence(s). A non-limiting list of
exemplary RSV viruses is provided in List 1.
[0440] In embodiments, the composition comprises at least 2, 3, 4,
5, 6, 7, 8, 9, 10 or even more different RNAs as defined in the
context of the first aspect of the invention each encoding at least
one peptide or protein derived from a genetically different RSV or
a fragment or variant thereof. The terms "different" or "different
RSV" as used throughout the present specification in the context of
a virus, e.g. "different" virus, has to be understood as the
difference between at least two respective viruses, wherein the
difference is manifested on the RNA genome of the respective
different virus. Particularly, said (genetically) different RSV
expresses at least one different protein, peptide or polyprotein,
wherein the at least one different protein, peptide or polyprotein
preferably differs in at least one amino acid.
[0441] In other embodiments, the composition comprises at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or even more further RNA constructs
encoding RSV antigens selected from glycoprotein G, short
hydrophobic protein SH, matrix protein M, nucleoprotein N, large
polymerase L, M2-1 protein, M2-2 protein, phosphoprotein P,
non-structural protein NS1 or non-structural protein NS2, or any
combination thereof.
[0442] For the production of a composition comprising at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 further RNA constructs encoding RSV,
methods as disclosed in the PCT application PCT/EP2016/082487 or in
published patent application WO2017/1090134A1 are preferably used
and adapted accordingly.
[0443] In preferred embodiments, the composition of the second
aspect comprises at least one artificial RNA of the first aspect
and at least one further artificial RNA comprising at least one
coding sequence encoding at least one antigenic peptide or protein
derived from RSV selected from matrix protein M, nucleoprotein N,
M2-1 protein, and/or phosphoprotein P or combinations thereof. The
addition of a further artificial RNA comprising at least one coding
sequence encoding at least one antigenic peptide or protein derived
from RSV M, N, M2-1, and/or phosphoprotein P (or combinations
thereof) is particularly advantageous to e.g. promote a T-cell
immune response.
[0444] Notably, embodiments relating to the artificial RNA of the
first aspect may likewise be read on and be understood as suitable
embodiments of the at least one further artificial RNA of the
second aspect (e.g., embodiments relating to UTR combinations, cds
optimizations, histone stem loop, PolyA, PolyC, cap structure, mRNA
structure, mRNA production and mRNA purification etc.).
[0445] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F and at least one further artificial RNA comprising
at least one coding sequence encoding at least one antigenic
peptide or protein derived from RSV matrix protein M.
[0446] Alternatively, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F and at least one further artificial RNA comprising
at least one coding sequence encoding at least one antigenic
peptide or protein derived from RSV N.
[0447] Alternatively, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F and at least one further artificial RNA comprising
at least one coding sequence encoding at least one antigenic
peptide or protein derived from RSV M2-1.
[0448] Alternatively, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F and at least one further artificial RNA comprising
at least one coding sequence encoding at least one antigenic
peptide or protein derived from RSV P.
[0449] In preferred embodiments, the composition of the second
aspect comprises two further artificial RNA species each comprising
at least one coding sequence encoding at least one antigenic
peptide or protein derived from RSV selected from matrix protein M,
nucleoprotein N, M2-1, and phosphoprotein P.
[0450] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M, and at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV N.
[0451] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV N, and at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV M2-1.
[0452] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV P, and at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV M2-1.
[0453] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV P, and at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV N.
[0454] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M, and at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV M2-1.
[0455] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M, and at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV P.
[0456] In preferred embodiments, the composition of the second
aspect comprises three further artificial RNA species each
comprising at comprising three further artificial RNA species each
comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV selected from matrix
protein M, nucleoprotein N, M2-1, and phosphoprotein P.
[0457] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M, at least one further artificial RNA
comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV N, and at least one
further artificial RNA comprising at least one coding sequence
encoding at least one antigenic peptide or protein derived from RSV
P.
[0458] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M2-1, at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV N, and at least one
further artificial RNA comprising at least one coding sequence
encoding at least one antigenic peptide or protein derived from RSV
P.
[0459] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M2-1, at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV M, and at least one
further artificial RNA comprising at least one coding sequence
encoding at least one antigenic peptide or protein derived from RSV
P.
[0460] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M2-1, at least one further artificial
RNA comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV M, and at least one
further artificial RNA comprising at least one coding sequence
encoding at least one antigenic peptide or protein derived from RSV
N.
[0461] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F and at least one further artificial RNA comprising
at least one coding sequence encoding at least one antigenic
peptide or protein derived from RSV M2-1.
[0462] In preferred embodiments, the composition of the second
aspect comprises four further artificial RNA species each
comprising at comprising three further artificial RNA species each
comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV selected from matrix
protein M, nucleoprotein N, M2-1, and phosphoprotein P.
[0463] Accordingly, the composition of the second aspect may
suitably comprise at least one artificial RNA of the first aspect
encoding RSF F, at least one further artificial RNA comprising at
least one coding sequence encoding at least one antigenic peptide
or protein derived from RSV M, at least one further artificial RNA
comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV N, at least one
further artificial RNA comprising at least one coding sequence
encoding at least one antigenic peptide or protein derived from RSV
P, and at least one further artificial RNA comprising at least one
coding sequence encoding at least one antigenic peptide or protein
derived from RSV M2-1.
[0464] In preferred embodiments of the second aspect, the coding
sequence of the further artificial RNA encodes at least one of the
amino acid sequences being identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to any one of SEQ ID NOs: 9684, 10053-10133, 10134,
10503-10636, 10637, 11006-11182, 11183, 11552-11725, 19844, 20213,
20582, 20951 or a fragment or variant of any of these sequences.
Additional information regarding each of these suitable amino acid
sequences encoding RSV proteins may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223> as explained in the following.
[0465] In further embodiments of the second aspect, the coding
sequence of the further artificial RNA encodes at least one
antigenic peptide or protein as defined herein and additionally a
heterologous secretory signal sequence or heterologous secretory
signal peptide. The heterologous secretory signal sequence may
increase the secretion of the encoded antigenic peptide or
protein.
[0466] Suitable secretory signal peptides may be selected from the
list of amino acid sequences according to SEQ ID NOs: 1-1115 and
SEQ ID NO: 1728 of the patent application WO2017/081082, or
fragments or variants of these sequences. On nucleic acid level,
any nucleic acid sequence (e.g. RNA sequence) may be selected which
encodes such amino acid sequences. In this context, the disclosure
of WO2017/081082 is herewith incorporated by reference.
[0467] According to embodiments, the secretory signal sequence
comprises an amino acid sequence being identical or at least 70%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical to any one of SEQ ID NOs: 21329-21362 or
a fragment or variant of any of these sequences. Additional
information regarding each of these suitable amino acid sequences
encoding secretory signal sequences may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223>.
[0468] In preferred embodiments, the further artificial RNA of the
composition comprises [0469] a) at least one heterologous 5'
untranslated region (5'-UTR) and/or at least one heterologous 3'
untranslated region (3'-UTR); and [0470] b) at least one coding
sequence operably linked to said 3'-UTR and/or 5'-UTR encoding at
least one antigenic peptide or protein derived from a RSV M, N,
M2-1, and/or phosphoprotein P or a fragment or variant thereof,
preferably encoding an amino acid sequence selected from any one of
SEQ ID NOs: 9684, 10053-10133, 10134, 10503-10636, 10637,
11006-11182, 11183, 11552-11725, 19844, 20213, 20582, 20951 or a
fragment or variant of any of these sequences.
[0471] In preferred embodiments, the further artificial RNA of the
composition comprises [0472] a) at least one heterologous 5'
untranslated region (5'-UTR) and/or at least one heterologous 3'
untranslated region (3'-UTR); and [0473] b) at least one coding
sequence operably linked to said 3'-UTR and/or 5'-UTR encoding at
least one antigenic peptide or protein derived from a RSV M2-1 or a
fragment or variant thereof, preferably encoding an amino acid
sequence selected from any one of SEQ ID NOs: 11183, 20953, 21414
or a fragment or variant of any of these sequences.
[0474] Suitably, the further artificial RNA of the composition
comprises a coding sequence is operably linked to a 3'-UTR and a
5'-UTR selected from a-1, a-2, a-3, a-4, a-5, b-1, b-2, b-3, b-4,
b-5, c-1, c-2, c-3, c-4, c-5, d-1, d-2, d-3, d-4, d-5, e-1, e-2,
e-3, e-4, e-5, e-6, f-1, f-2, f-3, f-4, f-5, g-1, g-2, g-3, g-4,
g-5, h-1, h-2, h- 3, h-4, h-5, i-1, i-2, or i-3 (as defined in the
context of the first aspect).
[0475] Suitably, the further artificial RNA of the composition
comprises a coding sequence wherein at least one or more than one,
or wherein preferably all uracil nucleotides are replaced by
pseudouridine (L) nucleotides or N1-methylpseudouridine (m1L)
nucleotides.
[0476] Accordingly, in other embodiments, the further artificial
RNA of of the composition may comprise a 5'-cap sequence element
according to SEQ ID NOs: 43 or 21321, or a fragment or variant
thereof.
[0477] In preferred embodiments of the second aspect, the coding
sequence of the further RNA comprises at least one of the nucleic
acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to SEQ ID NOs: 9685-9692, 10135-10142, 10638-10645,
11184-11119, 19845-19852, 20214-20221, 20583-20590, 20952-20959,
21385-21388, 21411-21414 or a fragment or a fragment or variant of
any of these sequences (cds optimizations as defined in the context
of the first aspect). Additional information regarding each of
these suitable amino acid sequences encoding RSV proteins may also
be derived from the sequence listing, in particular from the
details provided therein under identifier <223> as explained
in the following.
[0478] In preferred embodiments of the second aspect, the further
artificial RNA, preferably the further mRNA of the composition
comprises preferably in 5'- to 3-direction the following elements
a)-i): [0479] a) 5'-cap structure, preferably as specified in the
context of the first aspect, most preferably a Cap1 structure;
[0480] b) optionally, 5'-UTR as specified in the context of the
first aspect, preferably at least one selected from SEQ ID NOs:
1-22; [0481] c) a ribosome binding site, preferably as specified
herein; d) at least one coding sequence selected from SEQ ID NOs:
9685-9692, 10135-10142, 10638-10645, 11184-11119, 19845-19852,
20214-20221, 20583-20590, 20952-20959, 21385-21388, 21411-21414;
[0482] e) 3'-UTR as as specified in the context of the first
aspect, preferably at least one selected from SEQ ID NOs: 23-38;
[0483] f) optionally, a poly(A) sequence, preferably as specified
in the context of the first aspect; [0484] g) optionally, a poly(C)
sequence, preferably as specified in the context of the first
aspect; [0485] h) optionally, a histone stem-loop, preferably as
specified in the context of the first aspect; [0486] i) optionally,
a 3'-terminal sequence element as specified in the context of the
first aspect, preferably [0487] according to according to SEQ ID
NOs: 44-63, or 21322-21328, and wherein optionally at least one or
more than one, preferably wherein all uracil nucleotides are
replaced by pseudouridine (.psi.) nucleotides or
N1-methylpseudouridine (m1.psi.) nucleotides.
[0488] In particularly preferred embodiments of the second aspect,
the further artificial RNA, preferably the further mRNA of the
composition comprises preferably in 5'- to 3'-direction the
following elements a)-i): [0489] a) 5'-cap structure, preferably as
specified in the context of the first aspect, most preferably a
Cap1 structure; [0490] b) optionally, 5'-UTR as specified in the
context of the first aspect, preferably at least one selected from
SEQ ID NOs: 1-22; [0491] c) a ribosome binding site, preferably as
specified herein; [0492] d) at least one coding sequence selected
from SEQ ID NOs: 11185-11191, 21388, 20952-20959, 21414; [0493] e)
3'-UTR as as specified in the context of the first aspect,
preferably at least one selected from SEQ ID NOs: 23-38; [0494] f)
optionally, a poly(A) sequence, preferably as specified in the
context of the first aspect; [0495] g) optionally, a poly(C)
sequence, preferably as specified in the context of the first
aspect; [0496] h) optionally, a histone stem-loop, preferably as
specified in the context of the first aspect; [0497] i) optionally,
a 3'-terminal sequence element as specified in the context of the
first aspect, preferably according to according to SEQ ID NOs:
44-63, or 21322-21328, and [0498] wherein optionally at least one
or more than one, preferably wherein all uracil nucleotides are
replaced by pseudouridine (Ly) nucleotides or
N1-methylpseudouridine (m1Ly) nucleotides.
[0499] In that context, preferred RSV polypeptide, nucleic acid,
and mRNA sequences of the second aspect are provided in Table
7A.
[0500] In Table 7A, Columns A to D represent specific suitable
constructs of the second aspect derived from RSV protein, wherein
Column A provides suitable sequences for M, Column B provides
suitable sequences for N, Column C provides suitable sequences for
P, and Column D provides suitable sequences M2-1.
[0501] The protein names/designs are indicated in row 1 (Columns A
to D), the protein SEQ ID NOs as provided in the sequence listing
are in row 2 ("PRT"). Preferred protein SEQ ID NOs of HRSV(A2) are
provided in row 3 ("HRSV(A2) PRT"). The SEQ ID NOs of corresponding
coding sequences for each indicated HRSV(A2) protein are provided
in rows 4-11 ("cds wt", "cds opt1", "cds opt2", "cds opt3", "cds
opt4", "cds opt5", "cds opt6", "cds opt11", respectively). The SEQ
ID NOs of corresponding mRNA constructs comprising said coding
sequences and suitable 3'-UTRs and 5'-UTRs according to the
invention are provided in rows 12 to 55 ("mRNA design a-1" to "mRNA
design i-3" as specified herein).
[0502] Preferred protein SEQ ID NOs of HRSV(Memphis-37) are
provided in row 56 ("Memphis-37 PRT"). The SEQ ID NOs of
corresponding coding sequences for each indicated HRSV(Memphis-37)
protein are provided in rows 57-64 ("cds wt", "cds opt1", "cds
opt2", "cds opt3", "cds opt4", "cds opt5", "cds opt6", "cds opt11",
respectively).
[0503] The SEQ ID NOs of corresponding mRNA constructs comprising
said coding sequences and suitable 3'-UTRs and 5'-UTRs according to
the invention are provided in rows 65 to 108 ("mRNA design a-" to
"mRNA design i-3" as specified herein).
[0504] Further information e.g. regarding the type of coding
sequence comprised in the indicated mRNA constructs is provided in
the <223> identifier of the respective SEQ ID NO in the
sequence listing.
TABLE-US-00011 TABLE 7A Preferred further coding sequences and mRNA
constructs of the composition or vaccine A B C D 1 Strain
Description RSV M RSV N RSV P RSV M2-1 2 divers PRT 10053-10133
10503-10636 11006-11182 11552-11725 3 HRSV(A2) PRT 9684 10134 10637
11183 4 HRSV(A2) cds wt 9685 10135 10638 11184 5 HRSV(A2) CDS opt1
9686, 21385 10136, 21386 10639, 21387 11185, 21388 6 HRSV(A2) cds
opt2 9687 10137 10640 11186 7 HRSV(A2) cds opt3 9688 10138 10641
11187 8 HRSV(A2) cds opt4 9689 10139 10642 11188 9 HRSV(A2) cds
opt5 9690 10140 10643 11189 10 HRSV(A2) cds opt6 9691 10141 10644
11190 11 HRSV(A2) cds opt11 9692 10142 10645 11191 12 HRSV(A2) mRNA
Design a-1 9693-9700, 10143-10150, 10646-10653, 11192-11199, 21481,
21482, 21483, 21484, 21485, 21486, 21487, 21488, 21627, 21628
21629, 21630 21631, 21632 21633, 21634 13 HRSV(A2) mRNA Design a-2
9701-9708 10151-10158 10654-10661 11200-11207 14 HRSV(A2) mRNA
Design a-3 9709-9716 10159-10166 10662-10669 11208-11215 15
HRSV(A2) mRNA Design a-4 9717-9724 10167-10174 10670-10677
11216-11223 16 HRSV(A2) mRNA Design a-5 9725-9732 10175-10182
10678-10685 11224-11231 17 HRSV(A2) mRNA Design b-1 9733-9740
10183-10190 10686-10693 11232-11239 18 HRSV(A2) mRNA Design b-2
9741-9748 10191-10198 10694-10701 11240-11247 19 HRSV(A2) mRNA
Design b-3 9749-9756 10199-10206 10702-10709 11248-11255 20
HRSV(A2) mRNA Design b-4 9757-9764 10207-10214 10710-10717
11256-11263 21 HRSV(A2) mRNA Design b-5 9765-9772 10215-10222
10718-10725 11264-11271 22 HRSV(A2) mRNA Design c-1 9773-9780
10223-10230 10726-10733 11272-11279 23 HRSV(A2) mRNA Design c-2
9781-9788 10231-10238 10734-10741 11280-11287 24 HRSV(A2) mRNA
Design c-3 9789-9796 10239-10246 10742-10749 11288-11295 25
HRSV(A2) mRNA Design c-4 9797-9804 10247-10254 10750-10757
11296-11303 26 HRSV(A2) mRNA Design c-5 9805-9812 10255-10262
10758-10765 11304-11311 27 HRSV(A2) mRNA Design d-1 9813-9820
10263-10270 10766-10773 11312-11319 28 HRSV(A2) mRNA Design d-2
9821-9828 10271-10278 10774-10781 11320-11327 29 HRSV(A2) mRNA
Design d-3 9829-9836 10279-10286 10782-10789 11328-11335 30
HRSV(A2) mRNA Design d-4 9837-9844 10287-10294 10790-10797
11336-11343 31 HRSV(A2) mRNA Design d-5 9845-9852 10295-10302
10798-10805 11344-11351 32 HRSV(A2) mRNA Design e-1 9853-9860
10303-10310 10806-10813 11352-11359 33 HRSV(A2) mRNA Design e-2
9861-9868 10311-10318 10814-10821 11360-11367 34 HRSV(A2) mRNA
Design e-3 9869-9876 10319-10326 10822-10829 11368-11375 35
HRSV(A2) mRNA Design e-4 9877-9884 10327-10334 10830-10837
11376-11383 36 HRSV(A2) mRNA Design e-5 9885-9892 10335-10342
10838-10845 11384-11391 37 HRSV(A2) mRNA Design e-6 9893-9900
10343-10350 10846-10853 11392-11399 38 HRSV(A2) mRNA Design f-1
9901-9908 10351-10358 10854-10861 11400-11407 39 HRSV(A2) mRNA
Design f-2 9909-9916 10359-10366 10862-10869 11408-11415 40
HRSV(A2) mRNA Design f-3 9917-9924 10367-10374 10870-10877
11416-11423 41 HRSV(A2) mRNA Design f-4 9925-9932 10375-10382
10878-10885 11424-11431 42 HRSV(A2) mRNA Design f-5 9933-9940
10383-10390 10886-10893 11432-11439 43 HRSV(A2) mRNA Design g-1
9941-9948 10391-10398 10894-10901 11440-11447 44 HRSV(A2) mRNA
Design g-2 9949-9956 10399-10406 10902-10909 11448-11455 45
HRSV(A2) mRNA Design g-3 9957-9964 10407-10414 10910-10917
11456-11463 46 HRSV(A2) mRNA Design g-4 9965-9972 10415-10422
10918-10925 11464-11471 47 HRSV(A2) mRNA Design g-5 9973-9980
10423-10430 10926-10933 11472-11479 48 HRSV(A2) mRNA Design h-1
9981-9988 10431-10438 10934-10941 11480-11487 49 HRSV(A2) mRNA
Design h-2 9989-9996 10439-10446 10942-10949 11488-11495 50
HRSV(A2) mRNA Design h-3 9997-10004 10447-10454 10950-10957
11496-11503 51 HRSV(A2) mRNA Design h-4 10005-10012 10455-10462
10958-10965 11504-11511 52 HRSV(A2) mRNA Design h-5 10013-10020
10463-10470 10966-10973 11512-11519 53 HRSV(A2) mRNA Design i-1
10021-10028 10471-10478 10974-10981 11520-11527 54 HRSV(A2) mRNA
Design i-2 10029-10044 10479-10494 10982-10997 11528-11543 55
HRSV(A2) mRNA Design i-3 10045-10052 10495-10502 10998-11005
11544-11551 56 Memphis-37 PRT 19844 20213 20582 20951 57 Memphis-37
CDS wt 19845 20214 20583 20952 58 Memphis-37 CDS opt1 19846, 21411
20215, 21412 20584, 21413 20953, 21414 59 Memphis-37 CDS opt2 19847
20216 20585 20954 60 Memphis-37 CDS opt3 19848 20217 20586 20955 61
Memphis-37 CDS opt4 19849 20218 20587 20956 62 Memphis-37 CDS opt5
19850 20219 20588 20957 63 Memphis-37 CDS opt6 19851 20220 20589
20958 64 Memphis-37 CDS opt11 19852 20221 20590 20959 65 Memphis-37
mRNA Design a-1 19853-19860, 20222-20229, 20591-20598, 20960-20967,
21553, 21554, 21555, 21556, 21557, 21558, 21559, 21560, 21699,
21700 21701, 21702 21703, 21704 21705, 21706 66 Memphis-37 mRNA
Design a-2 19861-19868 20230-20237 20599-20606 20968-20975 67
Memphis-37 mRNA Design a-3 19869-19876 20238-20245 20607-20614
20976-20983 68 Memphis-37 mRNA Design a-4 19877-19884 20246-20253
20615-20622 20984-20991 69 Memphis-37 mRNA Design a-5 19885-19892
20254-20261 20623-20630 20992-20999 70 Memphis-37 mRNA Design b-1
19893-19900 20262-20269 20631-20638 21000-21007 71 Memphis-37 mRNA
Design b-2 19901-19908 20270-20277 20639-20646 21008-21015 72
Memphis-37 mRNA Design b-3 19909-19916 20278-20285 20647-20654
21016-21023 73 Memphis-37 mRNA Design b-4 19917-19924 20286-20293
20655-20662 21024-21031 74 Memphis-37 mRNA Design b-5 19925-19932
20294-20301 20663-20670 21032-21039 75 Memphis-37 mRNA Design c-1
19933-19940 20302-20309 20671-20678 21040-21047 76 Memphis-37 mRNA
Design c-2 19941-19948 20310-20317 20679-20686 21048-21055 77
Memphis-37 mRNA Design c-3 19949-19956 20318-20325 20687-20694
21056-21063 78 Memphis-37 mRNA Design c-4 19957-19964 20326-20333
20695-20702 21064-21071 79 Memphis-37 mRNA Design c-5 19965-19972
20334-20341 20703-20710 21072-21079 80 Memphis-37 mRNA Design d-1
19973-19980 20342-20349 20711-20718 21080-21087 81 Memphis-37 mRNA
Design d-2 19981-19988 20350-20357 20719-20726 21088-21095 82
Memphis-37 mRNA Design d-3 19989-19996 20358-20365 20727-20734
21096-21103 83 Memphis-37 mRNA Design d-4 19997-20004 20366-20373
20735-20742 21104-21111 84 Memphis-37 mRNA Design d-5 20005-20012
20374-20381 20743-20750 21112-21119 85 Memphis-37 mRNA Design e-1
20013-20020 20382-20389 20751-20758 21120-21127 86 Memphis-37 mRNA
Design e-2 20021-20028 20390-20397 20759-20766 21128-21135 87
Memphis-37 mRNA Design e-3 20029-20036 20398-20405 20767-20774
21136-21143 88 Memphis-37 mRNA Design e-4 20037-20044 20406-20413
20775-20782 21144-21151 89 Memphis-37 mRNA Design e-5 20045-20052
20414-20421 20783-20790 21152-21159 90 Memphis-37 mRNA Design e-6
20053-20060 20422-20429 20791-20798 21160-21167 91 Memphis-37 mRNA
Design f-1 20061-20068 20430-20437 20799-20806 21168-21175 92
Memphis-37 mRNA Design f-2 20069-20076 20438-20445 20807-20814
21176-21183 93 Memphis-37 mRNA Design f-3 20077-20084 20446-20453
20815-20822 21184-21191 94 Memphis-37 mRNA Design f-4 20085-20092
20454-20461 20823-20830 21192-21199 95 Memphis-37 mRNA Design f-5
20093-20100 20462-20469 20831-20838 21200-21207 96 Memphis-37 mRNA
Design g-1 20101-20108 20470-20477 20839-20846 21208-21215 97
Memphis-37 mRNA Design g-2 20109-20116 20478-20485 20847-20854
21216-21223 98 Memphis-37 mRNA Design g-3 20117-20124 20486-20493
20855-20862 21224-21231 99 Memphis-37 mRNA Design g-4 20125-20132
20494-20501 20863-20870 21232-21239 100 Memphis-37 mRNA Design g-5
20133-20140 20502-20509 20871-20878 21240-21247 101 Memphis-37 mRNA
Design h-1 20141-20148 20510-20517 20879-20886 21248-21255 102
Memphis-37 mRNA Design h-2 20149-20156 20518-20525 20887-20894
21256-21263 103 Memphis-37 mRNA Design h-3 20157-20164 20526-20533
20895-20902 21264-21271 104 Memphis-37 mRNA Design h-4 20165-20172
20534-20541 20903-20910 21272-21279 105 Memphis-37 mRNA Design h-5
20173-20180 20542-20549 20911-20918 21280-21287 106 Memphis-37 mRNA
Design i-1 20181-20188 20550-20557 20919-20926 21288-21295 107
Memphis-37 mRNA Design i-2 20189-20204 20558-20573 20927-20942
21296-21311 108 Memphis-37 mRNA Design i-3 20205-20212 20574-20581
20943-20950 21312-21319
[0505] Accordingly, in preferred embodiments, the composition of
the second aspect comprises at least one further artificial RNA
comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV selected from matrix
protein M, nucleoprotein N, M2-1 protein, phosphoprotein P, wherein
the further artificial RNA comprises or consists of an RNA sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from SEQ ID NOs: 9693-10052,
10143-10502, 10646-11005, 11192-11551, 19853-20212, 20222-20581,
20591-20950, 20960-21319, 21481-21488, 21627-21634, 21553-21560,
21699-21706 or a fragment or variant of any of these sequences,
wherein, optionally, at least one or more than one, or wherein all
uracil nucleotides are replaced by pseudouridine (.psi.)
nucleotides or N1-methylpseudouridine (m1 .psi.) nucleotides.
Additional information regarding each of these suitable amino acid
sequences encoding RSV proteins may also be derived from the
sequence listing, in particular from the details provided therein
under identifier <223> as explained in the following.
[0506] In particularly preferred embodiments of the second aspect,
the composition of the second aspect comprises [0507] at least one
artificial RNA which is identical or at least 70%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from SEQ ID NOs:
78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373,
2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587,
4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801,
6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094,
12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677,
14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570,
13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629,
17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474,
19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698
(encoding RSV F as defined in the first aspect) and a further
artificial RNA which is identical or at least 70%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 9693-10052, 19853-20212, 21481, 21482,
21627, 21628, 21553, 21554, 21699, 21700 (encoding RSV M); or
[0508] at least one artificial RNA which is identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected
from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004,
2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218,
4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432,
6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278,
11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308,
14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153,
13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260,
17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105,
19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552,
21635-21698 (encoding RSV F as defined in the first aspect) and a
further artificial RNA which is identical or at least 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 10143-10502, 20222-20581, 21483,
21484, 21629, 21630, 21555, 21556, 21701, 21702 (encoding RSV N);
or [0509] at least one artificial RNA which is identical or at
least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence
selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,
1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849,
3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063,
6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277,
8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201,
13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784,
15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891,
16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736,
18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626,
21489-21552, 21635-21698 (encoding RSV F as defined in the first
aspect) and at least one further artificial RNA which is identical
or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs:
10646-11005, 20591-20950, 21485, 21486, 21631, 21632, 21557, 21558,
21703, 21704 (encoding RSV P); or [0510] at least one artificial
RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to a nucleic acid sequence selected from SEQ ID NOs: 78-482,
493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742,
2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956,
4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,
7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,
12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,
15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,
16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,
18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,
21415-21480, 21561-21626, 21489-21552, 21635-21698 (encoding RSV F
as defined in the first aspect) and at least one further artificial
RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to a nucleic acid sequence selected from the group consisting of
SEQ ID NOs: 11192-11551, 20960-21319, 21487, 21488, 21633, 21634,
21559, 21560, 21705, 21706 (encoding RSV M2-1); or [0511] at least
one artificial RNA which is identical or at least 70%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical to a nucleic acid sequence selected from SEQ ID NOs:
78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373,
2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587,
4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801,
6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094,
12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677,
14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570,
13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629,
17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474,
19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698
(encoding RSV F as defined in the first aspect) and at least one
further artificial RNA which is identical or at least 70%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identical to a nucleic acid sequence selected from SEQ
ID NOs: 9693-10052, 19853-20212, 21481, 21482, 21627, 21628, 21553,
21554, 21699, 21700 (encoding RSV M) and a further artificial RNA
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a
nucleic acid sequence selected from SEQ ID NOs: 10646-11005,
20591-20950, 21485, 21486, 21631, 21632, 21557, 21558, 21703, 21704
(encoding RSV P); [0512] at least one artificial RNA which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from SEQ ID NOs: 78-482, 493-897, 907-1266,
1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,
3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694,
5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908,
7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832,
12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415,
15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522,
16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367,
18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480,
21561-21626, 21489-21552, 21635-21698 (encoding RSV F as defined in
the first aspect) and at least one further artificial RNA which is
identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic
acid sequence selected from SEQ ID NOs: 9693-10052, 19853-20212,
21481, 21482, 21627, 21628, 21553, 21554, 21699, 21700 (encoding
RSV M) and a further artificial RNA which is identical or at least
70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected
from SEQ ID NOs: 10646-11005, 20591-20950, 21485, 21486, 21631,
21632, 21557, 21558, 21703, 21704 (encoding RSV P) and a further
artificial RNA which is identical or at least 70%, 80%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to a nucleic acid sequence selected from SEQ ID NOs:
10143-10502, 20222-20581, 21483, 21484, 21629, 21630, 21555, 21556,
21701, 21702 (encoding RSV N), or [0513] at least one artificial
RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to a nucleic acid sequence selected from SEQ ID NOs: 78-86,
21415-21417, 21561-21563, 474-482, 11735-11742, 21489, 21490,
21635, 21636, 12087-12094, 493-501, 21418-21420, 21564-21566,
889-897, 12104-12111, 21491, 21492, 21637, 21638, 12456-12463,
907-914, 21421-21423, 21567-21569, 1259-1266, 12473-12480,
21493-21495, 21639-21641, 12825-12832, 1276-1283, 21424-21426,
21570-21572, 1628-1635, 12842-12849, 21496-21498, 21642-21644,
13194-13201, 1645-1652, 21433-21435, 21579-21581, 1997-2004,
13949-13956, 21505-21507, 21651-21653, 14301-14308, 2014-2021,
21436-21438, 21582-21584, 2366-2373, 14318-14325, 21508-21510,
21654-21656, 14670-14677, 2383-2390, 21439-21441, 21585-21587,
2735-2742, 14687-14694, 21511-21513, 21657-21659, 15039-15046,
2752-2759, 21442-21444, 21588-21590, 3104-3111, 15056-15063,
21514-21516, 21660-21662, 15408-15415, 3121-3128, 21445-21447,
21591-21593, 3473-3480, 15425-15432, 21517-21519, 21663-21665,
15777-15784, 3490-3497, 21448-21450, 21594-21596, 3842-3849,
15794-15801, 21520-21522, 21666-21668, 16146-16153, 3859-3866,
21427-21429, 21573-21575, 4211-4218, 13211-13218, 21499-21501,
21645-21647, 13563-13570, 4228-4235, 21430-21432, 21576-21578,
4580-4587, 13580-13587, 21502-21504, 21648-21650, 13932-13939,
4597-4604, 21451-21453, 21597-21599, 4949-4956, 16163-16170,
21523-21525, 21669-21671, 16515-16522, 4966-4973, 21454-21456,
21600-21602, 5318-5325, 16532-16539, 21526-21528, 21672-21674,
16884-16891, 5335-5342, 21457-21459, 21603-21605, 5687-5694,
16901-16908, 21529-21531, 21675-21677, 17253-17260, 5704-5711,
21460-21462, 21606-21608, 6056-6063, 17270-17277, 21532-21534,
21678-21680, 17622-17629 (encoding RSV F as defined in the first
aspect) and at least one further artificial RNA which is identical
or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NOs:
11192-11551, 20960-21319, 21487, 21488, 21633, 21634, 21559, 21560,
21705, 21706 (encoding RSV M2-1),
[0514] wherein, optionally, at least one or more than one, or
wherein all uracil nucleotides are replaced by pseudouridine
(.psi.) nucleotides or N1-methylpseudouridine (m1.psi.)
nucleotides.
[0515] In various embodiments, different combinations of RSV F (F0,
F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0,
F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2,
F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4,
F_DSCav1_mut5, F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6,
F_DSCav1_mut7, F-del_DSCav1_mut7, F_DSCav1_mut8, F-del_DSCav1_mut8)
RNA constructs and RSV T-cell antigen RNA constructs (RSV M, N,
M2-1, or P) are suitably comprised in the composition (as disclosed
in Table 7B; combinations 1-64). The combinations 49-64 are
preferred, combination 64 is particularly preferred.
TABLE-US-00012 TABLE 7B Suitable combinations of RNA constructs
encoding RSV F and RNA constructs encoding T-cell antigens SEQ ID
NO: T-cell antigen SEQ ID NO: Combination RSV F construct Protein
construct Protein 1 F0 68 M 9684 2 F-del 483 M 9684 3 F_DSCav1 898
M 9684 4 F-del_DSCav1 1267 M 9684 5 F_DSCav1_mut1 1636 M 9684 6
F-del_DSCav1_mut1 2005 M 9684 7 F_DSCav1_mut2 2374 M 9684 8
F-del_DSCav1_mut2 2743 M 9684 9 F_DSCav1_mut3 3112 M 9684 10
F-del_DSCav1_mut3 3481 M 9684 11 F_DSCav1_mut0 3850 M 9684 12
F-del_DSCav1_mut0 4219 M 9684 13 F_DSCav1_mut4 4588 M 9684 14
F-del_DSCav1_mut4 4957 M 9684 15 F_DSCav1_mut5 5326 M 9684 16
F-del_DSCav1_mut5 5695 M 9684 17 F0 68 N 10134 18 F-del 483 N 10134
19 F_DSCav1 898 N 10134 20 F-del_DSCav1 1267 N 10134 21
F_DSCav1_mut1 1636 N 10134 22 F-del_DSCav1_mut1 2005 N 10134 23
F_DSCav1_mut2 2374 N 10134 24 F-del_DSCav1_mut2 2743 N 10134 25
F_DSCav1_mut3 3112 N 10134 26 F-del_DSCav1_mut3 3481 N 10134 27
F_DSCav1_mut0 3850 N 10134 28 F-del_DSCav1_mut0 4219 N 10134 29
F_DSCav1_mut4 4588 N 10134 30 F-del_DSCav1_mut4 4957 N 10134 31
F_DSCav1_mut5 5326 N 10134 32 F-del_DSCav1_mut5 5695 N 10134 33 F0
68 P 10637 34 F-del 483 P 10637 35 F_DSCav1 898 P 10637 36
F-del_DSCav1 1267 P 10637 37 F_DSCav1_mut1 1636 P 10637 38
F-del_DSCav1_mut1 2005 P 10637 39 F_DSCav1_mut2 2374 P 10637 40
F-del_DSCav1_mut2 2743 P 10637 41 F_DSCav1_mut3 3112 P 10637 42
F-del_DSCav1_mut3 3481 P 10637 43 F_DSCav1_mut0 3850 P 10637 44
F-del_DSCav1_mut0 4219 P 10637 45 F_DSCav1_mut4 4588 P 10637 46
F-del_DSCav1_mut4 4957 P 10637 47 F_DSCav1_mut5 5326 P 10637 48
F-del_DSCav1_mut5 5695 P 10637 49 F0 68 M2-1 11183 50 F-del 483
M2-1 11183 51 F_DSCav1 898 M2-1 11183 52 F-del_DSCav1 1267 M2-1
11183 53 F_DSCav1_mut1 1636 M2-1 11183 54 F-del_DSCav1_mut1 2005
M2-1 11183 55 F_DSCav1_mut2 2374 M2-1 11183 56 F-del_DSCav1_mut2
2743 M2-1 11183 57 F_DSCav1_mut3 3112 M2-1 11183 58
F-del_DSCav1_mut3 3481 M2-1 11183 59 F_DSCav1_mut0 3850 M2-1 11183
60 F-del_DSCav1_mut0 4219 M2-1 11183 61 F_DSCav1_mut4 4588 M2-1
11183 62 F-del_DSCav1_mut4 4957 M2-1 11183 63 F_DSCav1_mut5 5326
M2-1 11183 64 F-del_DSCav1_mut5 5695 M2-1 11183
[0516] Table 7B discloses compositions of the second aspect
comprising at least one RNA encoding RSV F from the strain A2 as
defined in the first aspect and at least one further artificial RNA
comprising at least one coding sequence encoding at least one
antigenic peptide or protein derived from RSV from the strain A2
selected from matrix protein M, nucleoprotein N, M2-1 protein,
phosphoprotein P. In further embodiments of the invention all
disclosed compositions in Table 7B are also applicable for
antigenic peptides or proteins derived from a RSV isolate
Memphis-37 (strain Memphis-37).
[0517] In a particularly preferred embodiment, the composition
comprises one RSV F RNA construct (F0, F-del, F0_DSCav1,
F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut1,
F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2, F_DSCav1_mut3,
F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4, F_DSCav1_mut5,
F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6, F_DSCav1_mut7,
F-del_DSCav1_mut7, F_DSCav1_mut8, F-del_DSCav1_mut8) and, in
addition, one RSV M2-1 RNA construct, preferably F-del_DSCav1_mut5
and M2-1.
[0518] In particularly preferred embodiments, one RNA construct
encoding RSV F, selected from SEQ ID NOs: 5704-5711, 21460-21462,
21606-21608, 17270-17277, 21532-21534, 21678-21680 and one RNA
construct encoding M2-1, selected from SEQ ID NOs: 11192-11199,
21487, 21488, 21633, 21634, 20960-20967, 21559, 21560, 21705, 21706
are comprised in the composition of the invention.
[0519] In various embodiments, the at least one artificial RNA of
the first aspect and the at least one further artificial RNA as
specified herein are derived from the same RSV virus (e.g. any
virus selected from List 1).
[0520] In preferred embodiments, the at least one artificial RNA of
the first aspect is derived from HRSV(A2) and the at least one
further artificial RNA as specified herein is derived from
HRSV(A2).
[0521] In preferred embodiments, the at least one artificial RNA of
the first aspect is derived from HRSV(Memphis-37) and the at least
one further artificial RNA as specified herein is derived from
HRSV(Memphis-37).
[0522] It has to be understood that in the context of the
invention, certain combinations of coding sequences may be
generated by any combination of monocistronic, bicistronic and
multicistronic artificial nucleic acids and/or
multi-antigen-constructs/nucleic acid to obtain a nucleic acid
composition encoding multiple antigenic peptides or proteins as
defined herein.
[0523] Furthermore, one or more compatible solid or liquid fillers
or diluents or encapsulating compounds may be used as well, which
are suitable for administration to a person. The term "compatible"
as used herein means that the constituents of the composition are
capable of being mixed with the at least one RNA and, optionally,
the further artificial RNA of the composition, in such a manner
that no interaction occurs, which would substantially reduce the
biological activity or the pharmaceutical effectiveness of the
composition under typical use conditions. Pharmaceutically
acceptable carriers, fillers and diluents must have sufficiently
high purity and sufficiently low toxicity to make them suitable for
administration to a person to be treated. Compounds which may be
used as pharmaceutically acceptable carriers, fillers or
constituents thereof are sugars, such as, for example, lactose,
glucose, trehalose and sucrose; starches, such as, for example,
corn starch or potato starch; dextrose; cellulose and its
derivatives, such as, for example, sodium carboxymethylcellulose,
ethylcellulose, cellulose acetate; powdered tragacanth; malt;
gelatin; tallow; solid glidants, such as, for example, stearic
acid, magnesium stearate; calcium sulfate; vegetable oils, such as,
for example, groundnut oil, cottonseed oil, sesame oil, olive oil,
corn oil and oil from theobroma; polyols, such as, for example,
polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene
glycol; alginic acid.
[0524] Further additives, which may be included in the composition
are emulsifiers, such as, for example, Tween; wetting agents, such
as, for example, sodium lauryl sulfate; colouring agents;
taste-imparting agents, pharmaceutical carriers; tablet-forming
agents; stabilizers; antioxidants; preservatives.
[0525] Complexation:
[0526] In a preferred embodiment of the second aspect, the at least
one artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed or associated
with or at least partially complexed or partially associated with
one or more cationic or polycationic compound, preferably cationic
or polycationic polymer, cationic or polycationic polysaccharide,
cationic or polycationic lipid, cationic or polycationic protein,
cationic or polycationic peptide, or any combinations thereof.
[0527] The term "cationic or polycationic compound" as used herein
will be recognized and understood by the person of ordinary skill
in the art, and are for example intended to refer to a charged
molecule, which is positively charged at a pH value ranging from
about 1 to 9, at a pH value ranging from about 3 to 8, at a pH
value ranging from about 4 to 8, at a pH value ranging from about 5
to 8, more preferably at a pH value ranging from about 6 to 8, even
more preferably at a pH value ranging from about 7 to 8, most
preferably at a physiological pH, e.g. ranging from about 7.2 to
about 7.5. Accordingly, a cationic component, e.g. a cationic
peptide, cationic protein, cationic polymer, cationic
polysaccharide, cationic lipid may be any positively charged
compound or polymer which is positively charged under physiological
conditions. A "cationic or polycationic peptide or protein" may
contain at least one positively charged amino acid, or more than
one positively charged amino acid, e.g. selected from Arg, His, Lys
or Orn. Accordingly, "polycationic" components are also within the
scope exhibiting more than one positive charge under the given
conditions.
[0528] Cationic or polycationic compounds, being particularly
preferred in this context may be selected from the following list
of cationic or polycationic peptides or proteins of fragments
thereof: protamine, nucleoline, spermine or spermidine, or other
cationic peptides or proteins, such as poly-L-lysine (PLL),
poly-arginine, basic polypeptides, cell penetrating peptides
(CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),
Tat-derived peptides, Penetratin, VP22 derived or analog peptides,
HSV VP22 (Herpes simplex), MAP, KALA or protein transduction
domains (PTDs), PpT620, prolin-rich peptides, arginine-rich
peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,
Calcitonin peptide(s), Antennapedia-derived peptides, pAntp, ps,
FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1),
pVEC, hCT-derived peptides, SAP, or histones. More preferably, the
nucleic acid as defined herein, preferably the mRNA as defined
herein, is complexed with one or more polycations, preferably with
protamine or oligofectamine, most preferably with protamine.
[0529] In a preferred embodiment of the second aspect, the at least
one artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with
protamine
[0530] Further preferred cationic or polycationic compounds, which
can be used as transfection or complexation agent may include
cationic polysaccharides, for example chitosan, polybrene etc.;
cationic lipids, e.g. DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT,
DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl
phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS, DIMRI,
DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9, oligofectamine; or cationic or
polycationic polymers, e.g. modified polyaminoacids, such as
beta-aminoacid-polymers or reversed polyamides, etc., modified
polyethylenes, such as PVP etc., modified acrylates, such as
pDMAEMA etc., modified amidoamines such as pAMAM etc., modified
polybetaaminoester (PBAE), such as diamine end modified 1,4
butanediol diacrylate-co-5-amino-1-pentanol polymers, etc.,
dendrimers, such as polypropylamine dendrimers or pAMAM based
dendrimers, etc., polyimine(s), such as PEI, poly(propyleneimine),
etc., polyallylamine, sugar backbone based polymers, such as
cyclodextrin based polymers, dextran based polymers, etc., silan
backbone based polymers, such as PMOXA-PDMS copolymers, etc.,
blockpolymers consisting of a combination of one or more cationic
blocks (e.g. selected from a cationic polymer as mentioned above)
and of one or more hydrophilic or hydrophobic blocks (e.g.
polyethyleneglycole); etc.
[0531] In this context it is particularly preferred that the at
least one artificial RNA as defined herein and, optionally, the
further artificial RNA of the second aspect, is complexed or at
least partially complexed with a cationic or polycationic compound
and/or a polymeric carrier, preferably cationic proteins or
peptides. In this context, the disclosure of WO2010/037539 and
WO2012/113513 is incorporated herewith by reference. Partially
means that only a part of the artificial nucleic acid is complexed
with a cationic compound and that the rest of the artificial
nucleic acid is (comprised in the inventive (pharmaceutical)
composition) in uncomplexed form ("free").
[0532] In a preferred embodiment of the second aspect, the at least
one artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
cationic or polycationic compounds, preferably protamine, and at
least one free artificial RNA of the first aspect and, optionally
the further RNA of the second aspect.
[0533] In this context it is particularly preferred that the at
least one artificial RNA as defined herein, and, optionally, the
further artificial RNA of the second aspect, is complexed, or at
least partially complexed with protamine.
[0534] Preferably, the molar ratio of the nucleic acid,
particularly the RNA of the protamine-complexed RNA to the free RNA
may be selected from a molar ratio of about 0.001:1 to about
1:0.001, including a ratio of about 1:1. Suitably, the complexed
RNA is complexed with protamine by addition of protamine-trehalose
solution to the RNA sample at a RNA:protamine weight to weight
ratio (w/w) of 2:1.
[0535] Further preferred cationic or polycationic proteins or
peptides that may be used for complexation can be derived from
formula (Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x of the patent
application WO2009/030481 or WO2011/026641, the disclosure of
WO2009/030481 or WO2011/026641 relating thereto incorporated
herewith by reference.
[0536] In a preferred embodiment of the second aspect, the at least
one artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed, or at least
partially complexed with at least one cationic or polycationic
proteins or peptides preferably selected from SEQ ID NOs: 64-67,
21320 or any combinations thereof.
[0537] According to embodiments, the composition of the present
invention comprises the RNA as defined herein, and a polymeric
carrier.
[0538] The term "polymeric carrier" as used herein will be
recognized and understood by the person of ordinary skill in the
art, and are for example intended to refer to a compound that
facilitates transport and/or complexation of another compound
(cargo). A polymeric carrier is typically a carrier that is formed
of a polymer. A polymeric carrier may be associated to its cargo
(nucleic acid, RNA) by covalent or non-covalent interaction.
[0539] A suitable polymeric carrier may be a polymeric carrier
formed by disulfide-crosslinked cationic compounds. The
disulfide-crosslinked cationic compounds may be the same or
different from each other. The polymeric carrier can also contain
further components. The polymeric carrier used according to the
present invention may comprise mixtures of cationic peptides,
proteins or polymers and optionally further components as defined
herein, which are crosslinked by disulfide bonds (via --SH
groups).
[0540] In this context, polymeric carriers according to formula
{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa')x(Cys)y} and formula
Cys,{(Arg);(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys2 of the patent
application WO2012/013326 are preferred, the disclosure of
WO2012/013326 relating thereto incorporated herewith by
reference.
[0541] In embodiments, the polymeric carrier used to complex the
RNA as defined herein may be derived from a polymeric carrier
molecule according formula
(L-P.sup.1-S-[S-P.sup.2-S].sub.n-S-P.sup.1-L) of the patent
application WO2011/026641, the disclosure of WO2011/026641 relating
thereto incorporated herewith by reference.
[0542] In embodiments, the polymeric carrier compound is formed by,
or comprises or consists of the peptide elements CysArg12Cys (SEQ
ID NO: 64) or CysArg12 (SEQ ID NO: 65) or TrpArg12Cys (SEQ ID NO:
66). In particularly preferred embodiments, the polymeric carrier
compound consists of a (R.sub.12C)-(R.sub.12C) dimer, a
(WR.sub.12C)-(WR.sub.12C) dimer, or a
(CR.sub.12)-(CR.sub.12C)-(CR.sub.12) trimer, wherein the individual
peptide elements in the dimer (e.g. (WR.sub.12C)), or the trimer
(e.g. (CR.sub.12)), are connected via --SH groups.
[0543] In a preferred embodiment of the second aspect, the at least
one artificial RNA of the first aspect and optionally the further
artificial RNA of the second aspect, is complexed or associated
with a polyethylene glycol/peptide polymer comprising
HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)7-S-PEG5000-OH (SEQ ID NO: 67
as peptide monomer).
[0544] In a further preferred embodiment of the second aspect, the
at least one artificial RNA of the first aspect and, optionally,
the further artificial RNA of the second aspect, is complexed or
associated with a polyethylene glycol/peptide polymer comprising
HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)4-S-PEG5000-OH (SEQ ID NO: 67
as peptide monomer).
[0545] In a further preferred embodiment of the second aspect, the
at least one artificial RNA of the first aspect and, optionally,
the further artificial RNA of the second aspect, is complexed or
associated with a polyethylene glycol/peptide polymer comprising
HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)7-S-PEG5000-OH (SEQ ID NO:
21320 as peptide monomer).
[0546] In a further preferred embodiment of the second aspect, the
at least one artificial RNA of the first aspect and, optionally,
the further artificial RNA of the second aspect, is complexed or
associated with a polyethylene glycol/peptide polymer comprising
HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)4-S-PEG5000-OH (SEQ ID NO:
21320 as peptide monomer).
[0547] In other embodiments, the composition comprises at least one
artificial RNA as described herein, and, optionally, the further
artificial RNA of the second aspect, wherein the at least one
artificial RNA and, optionally, the further artificial RNA of the
second aspect, is complexed or associated with polymeric carriers
and, optionally, with at least one lipid component as described in
the published PCT applications WO2017/212008A1, WO2017/212006A1,
WO2017/212007A1, and WO2017/212009A1. In this context, the
disclosures of WO2017/212008A1, WO2017/212006A1, WO2017/212007A1,
and WO2017/212009A1 are herewith incorporated by reference.
[0548] In a particularly preferred embodiment, the polymeric
carrier is a peptide polymer, preferably a polyethylene
glycol/peptide polymer as defined above, and a lipid component,
preferably a lipidoid component, more preferably lipidoid
component.
[0549] In preferred embodiment of the second aspect, the at least
one artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed or associated
with a polymeric carrier, preferably with a polyethylene
glycol/peptide polymer as defined above, and a lipidoid component,
wherein the lipidoid component is a compound according to formula
A
##STR00001##
[0550] wherein [0551] R.sub.A is independently selected for each
occurrence an unsubstituted, cyclic or acyclic, branched or
unbranched C.sub.1-20 aliphatic group; a substituted or
unsubstituted, cyclic or acyclic, branched or unbranched C.sub.1-20
heteroaliphatic group; a substituted or unsubstituted aryl; a
substituted or unsubstituted heteroaryl;
##STR00002##
[0552] wherein at least one R.sub.A is
##STR00003## [0553] R.sub.5 is independently selected for each
occurrence of from an unsubstituted, cyclic or acyclic, branched or
unbranched C.sub.8-16 aliphatic; a substituted or unsubstituted
aryl; or a substituted or unsubstituted heteroaryl; [0554] each
occurrence of x is an integer from 1 to 10; [0555] each occurrence
of y is an integer from 1 to 10;
[0556] or a pharmaceutically acceptable salt thereof.
[0557] In a preferred embodiment, the lipidoid component is
3-C12-OH according to formula B
##STR00004##
[0558] In preferred embodiments, the peptide polymer comprising
lipidoid 3-C12-OH as specified above is used to complex the RNA of
the first aspect and, optionally, the further artificial RNA of the
second aspect, to form complexes having an N/P ratio from about 0.1
to about 20, or from about 0.2 to about 15, or from about 2 to
about 15, or from about 2 to about 12, wherein the N/P ratio is
defined as the mole ratio of the nitrogen atoms of the basic groups
of the cationic peptide or polymer to the phosphate groups of the
artificial nucleic acid. In that context, the disclosure of
WO2017/212009A1, in particular Claims 1 to 10 of WO2017/212009A1,
and the specific disclosure relating thereto is herewith
incorporated by reference.
[0559] Encapsulation/Complexation in LNPs:
[0560] In preferred embodiments of the second aspect, the
artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed or associated
with one or more lipids (e.g. cationic lipids and/or neutral
lipids), thereby forming liposomes, lipid nanoparticles (LNPs),
lipoplexes, and/or nanoliposomes.
[0561] For compositions comprising more than one artificial RNA
construct as defined herein (e,g, F-del and M2-1), said constructs
may be co-formulated in e.g. LNPs to form the respective
composition.
[0562] Alternatively, said more than one artificial RNA constructs
may be formulated separately, and may subsequently be combined, to
form the respective composition.
[0563] In this context, the terms "complexed" or "associated" refer
to the essentially stable combination of artificial RNA of the
first aspect and, optionally, the further artificial RNA of the
second aspect, with one or more lipids into larger complexes or
assemblies without covalent binding.
[0564] The term "lipid nanoparticle", also referred to as "LNP", is
not restricted to any particular morphology, and include any
morphology generated when a cationic lipid and optionally one or
more further lipids are combined, e.g. in an aqueous environment
and/or in the presence of RNA. For example, a liposome, a lipid
complex, a lipoplex and the like are within the scope of a lipid
nanoparticle (LNP).
[0565] Accordingly, in preferred embodiments of the second aspect,
the artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
lipids thereby forming lipid nanoparticles (LNP).
[0566] LNPs typically comprise a cationic lipid and one or more
excipient selected from neutral lipids, charged lipids, steroids
and polymer conjugated lipids (e.g. PEGylated lipid). The RNA may
be encapsulated in the lipid portion of the LNP or an aqueous space
enveloped by some or the entire lipid portion of the LNP. The RNA
or a portion thereof may also be associated and complexed with the
LNP. An LNP may comprise any lipid capable of forming a particle to
which the nucleic acids are attached, or in which the one or more
nucleic acids are encapsulated.
[0567] Preferably, the LNP comprising nucleic acids comprises one
or more cationic lipids, and one or more stabilizing lipids.
Stabilizing lipids include neutral lipids and PEGylated lipids.
[0568] The cationic lipid of an LNP may be cationisable, i.e. it
becomes protonated as the pH is lowered below the pK of the
ionizable group of the lipid, but is progressively more neutral at
higher pH values. At pH values below the pK, the lipid is then able
to associate with negatively charged nucleic acids. In certain
embodiments, the cationic lipid comprises a zwitterionic lipid that
assumes a positive charge on pH decrease.
[0569] The LNP may comprise any further cationic or cationisable
lipid, i.e. any of a number of lipid species which carry a net
positive charge at a selective pH, such as physiological pH.
[0570] Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
1,2-dioleoyltrimethyl ammonium propane chloride (DOTAP) (also known
as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and
1,2-Dioleyloxy-3-trimethylaminopropane chloride salt),
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (.gamma.-DLenDMA),
1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.CI), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.CI), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),
1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM
A), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA) or analogs thereof,
(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-
-3aH-cyclopenta[d][1,3]dioxol-5-amine,
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)b-
utanoate (MC3),
1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami-
no)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA),
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino) butanoate (DLin-M-C3-DMA),
3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethy-
lpropan-1-amine (MC3 Ether),
4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethy-
lbutan-1-amine (MC4 Ether), LIPOFECTIN.RTM. (commercially available
cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand
Island, N.Y.); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising
N-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic lipids comprising
dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from
Promega Corp., Madison, Wis.) or any combination of any of the
foregoing.
[0571] In some embodiments, the lipid is selected from the group
consisting of 98N12-5, C12-200, and ckk-E12.
[0572] In one embodiment, the further cationic lipid is an amino
lipid.
[0573] Representative amino lipids include, but are not limited to,
1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-dilinoleyoxy-3morpholinopropane (DLin-MA),
1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP),
1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.CI), 1,2-dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.CI), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), 3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-dioleylamino)-1,2-propanediol (DOAP),
1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), and
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate
(DLin-MC3-DMA); MC3 (US20100324120).
[0574] In one embodiment, the artificial RNA of the first aspect
and, optionally, the further artificial RNA of the second aspect,
may be formulated in an aminoalcohol lipidoid. Aminoalcohol
lipidoids which may be used in the present invention may be
prepared by the methods described in U.S. Pat. No. 8,450,298,
herein incorporated by reference in its entirety. Suitable
(ionizable) lipids can also be the compounds as disclosed in Tables
1, 2 and 3 and as defined in claims 1-24 of WO2017/075531A1, hereby
incorporated by reference.
[0575] In another embodiment, ionizable lipids can also be the
compounds as disclosed in WO2015/074085A1 (i.e. ATX-001 to ATX-032
or the compounds as specified in claims 1-26), U.S. Appl. Nos.
61/905,724 and 15/614,499 or U.S. Pat. Nos. 9,593,077 and 9,567,296
hereby incorporated by reference in their entirety.
[0576] In that context, any lipid derived from generic formula
(X1)
##STR00005##
[0577] wherein, Ri and R2 are the same or different, each a linear
or branched alkyl consisting of 1 to 9 carbons, an alkenyl or
alkynyl consisting of 2 to 11 carbons, Li and L2 are the same or
different, each a linear alkylene or alkenylene consisting of 5 to
18 carbons, or forming a heterocycle with N, Xi is a bond, or is
--CO--O-- whereby -L2-CO--O--R2 is formed, X2 is S or O, L3 is a
bond or a linear or branched alkylene consisting of 1 to 6 carbons,
or forming a heterocycle with N, R3 is a linear or branched
alkylene consisting of 1 to 6 carbons, and R4 and R 5 are the same
or different, each hydrogen or a linear or branched alkyl
consisting of 1 to 6 carbons; or a pharmaceutically acceptable salt
thereof may be suitably used.
[0578] In other embodiments, suitable cationic lipids can also be
the compounds as disclosed in WO2017/117530A1 (i.e. lipids 13, 14,
15, 16, 17, 18, 19, 20, or the compounds as specified in the
claims), hereby incorporated by reference in its entirety.
[0579] In that context, any lipid derived from generic formula
(X2)
##STR00006##
[0580] wherein
[0581] X is a linear or branched alkylene or alkenylene,
monocyclic, bicyclic, or tricyclic arene or heteroarene;
[0582] Y is a bond, an ethene, or an unsubstituted or substituted
aromatic or heteroaromatic ring; Z is S or O;
[0583] L is a linear or branched alkylene of 1 to 6 carbons;
[0584] R-3 and R4 are independently a linear or branched alkyl of 1
to 6 carbons;
[0585] Ri and R2 are independently a linear or branched alkyl or
alkenyl of 1 to 20 carbons; r is 0 to 6; and
[0586] m, n, p, and q are independently 1 to 18;
[0587] wherein when n=q, m=p, and Ri=R2, then X and Y differ;
[0588] wherein when X=Y, n=q, m=p, then Ri and R2 differ;
[0589] wherein when X=Y, n=q, and Ri=R2, then m and p differ;
and
[0590] wherein when X=Y, m=p, and Ri=R2, then n and q differ;
[0591] or a pharmaceutically acceptable salt thereof.
[0592] In preferred embodiments, a lipid may be used derived from
formula (X2), wherein, X is a bond, linear or branched alkylene,
alkenylene, or monocyclic, bicyclic, or tricyclic arene or
heteroarene; Y is a monocyclic, bicyclic, or tricyclic arene or
heteroarene; Z is S or O; L is a linear or branched alkylene of 1
to 6 carbons; R3 and R4 are independently a linear or branched
alkyl of 1 to 6 carbons; Ri and R2 are independently a linear or
branched alkyl or alkenyl of 1 to 20 carbons; r is 0 to 6; and m,
n, p, and q are independently 1 to 18; or a pharmaceutically
acceptable salt thereof may be suitably used.
[0593] In preferred embodiments, ionizable lipids may also be
selected from the lipid compounds disclosed in PCT application
PCT/EP2017/077517 (i.e. lipid compounds derived form formula I, II,
and III of PCT/EP2017/077517, or lipid compounds as specified in
Claims 1 to 12 of PCT/EP2017/077517), the disclosure of
PCT/EP2017/077517 hereby incorporated by reference in its entirety.
In that context, lipid compounds disclosed in Table 7 of
PCT/EP2017/077517 (e.g. lipid compounds derived from formula I-1 to
I-41) and lipid compounds disclosed in Table 8 of PCT/EP2017/077517
(e.g. lipid compounds derived from formula I-1 to II-36) may be
suitably used in the context of the invention. Accordingly, formula
I-1 to formula I-41 and formula II-1 to formula I-36 of
PCT/EP2017/077517, and the specific disclosure relating thereto,
are herewith incorporated by reference.
[0594] In particularly preferred embodiments of the second aspect,
a suitable lipid may be a cationic lipid according to formula
(III)
##STR00007##
[0595] or a pharmaceutically acceptable salt, tautomer, prodrug or
stereoisomer thereof, wherein, R1, R2, R3, L1, L2, G1, G2, and G3
are as below.
[0596] Formula (III) is further defined in that:
[0597] one of L.sup.1 or L.sup.2 is --O(C.dbd.O)--, --(C.dbd.O)O--,
--C(.dbd.O)--, --O--, --S(O)--, --S--S--, --C(.dbd.O)S--,
SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
--NR.sup.aC(.dbd.O)NR.sup.a--, --OC(.dbd.O)NR.sup.a-- or
--NR.sup.aC(.dbd.O)O--, and the other of L1 or L.sup.2 is
--O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O)--,
--S--S--, --C(.dbd.O)S--, SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)NR.sup.a--,
--OC(.dbd.O)NR.sup.a-- or --NR.sup.aC(.dbd.O)O-- or a direct
bond;
[0598] G.sup.1 and G.sup.2 are each independently unsubstituted
C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene;
[0599] G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24
alkenylene, C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.8
cycloalkenylene;
[0600] R.sup.a is H or C.sub.1-C.sub.12 alkyl;
[0601] R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24
alkyl or C.sub.6-C.sub.24 alkenyl;
[0602] R.sup.3 is H, OR.sup.5, CN, --C(.dbd.O)OR.sup.4,
--OC(.dbd.O)R.sup.4 or --NR.sup.5C(.dbd.O)R.sup.4;
[0603] R.sup.4 is C.sub.1-C.sub.12 alkyl;
[0604] R.sup.5 is H or C.sub.1-C.sub.12 alkyl; and
[0605] x is 0, 1 or 2.
[0606] In some of the foregoing embodiments of formula (III), the
lipid has one of the following structures (IIIA) or (IIIB):
##STR00008##
[0607] wherein:
[0608] A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;
R.sup.6 is, at each occurrence, independently H, OH or
C.sub.1-C.sub.24 alkyl; n is an integer ranging from 1 to 15.
[0609] In some of the foregoing embodiments of formula (III), the
lipid has structure (IIIA), and in other embodiments, the lipid has
structure (IIIB).
[0610] In other embodiments of formula (III), the lipid has one of
the following structures (IIC) or (IIID):
##STR00009##
[0611] wherein y and z are each independently integers ranging from
1 to 12.
[0612] In any of the foregoing embodiments of formula (III), one of
L.sup.1 or L.sup.2 is --O(C.dbd.O)--. For example, in some
embodiments each of L.sup.1 and L.sup.2 are --O(C.dbd.O)--. In some
different embodiments of any of the foregoing, L.sup.1 and L.sup.2
are each independently --(C.dbd.O)O-- or --O(C.dbd.O)--. For
example, in some embodiments each of L.sup.1 and L.sup.2 is
--(C.dbd.O)O--.
[0613] In preferred embodiments of the second aspect, the cationic
lipid of the LNP is a compound of formula I, wherein:
[0614] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)-- or
(C.dbd.O)--O--;
[0615] G.sup.3 is C.sub.1-C.sub.24 alkylene or C.sub.1-C.sub.24
alkenylene; and
[0616] R.sup.3 is H or OR.sup.5.
[0617] In some different embodiments of formula (III), the lipid
has one of the following structures (IIIE) or (IIIF):
##STR00010##
[0618] In some of the foregoing embodiments of formula (III), the
lipid has one of the following structures (IIIG), (IIIH),
##STR00011##
[0619] In some of the foregoing embodiments of formula (III), n is
an integer ranging from 2 to 12, for example from 2 to 8 or from 2
to 4. In some embodiments, n is 3, 4, 5 or 6. In some embodiments,
n is 3. In some embodiments, n is 4. In some embodiments, n is 5.
In some embodiments, n is 6. In some other of the foregoing
embodiments of formula (III), y and z are each independently an
integer ranging from 2 to 10. For example, in some embodiments, y
and z are each independently an integer ranging from 4 to 9 or from
4 to 6. In some of the foregoing embodiments of formula (II),
R.sup.6 is H. In other of the foregoing embodiments, R.sup.6 is
C.sub.1-C.sub.24 alkyl. In other embodiments, R.sup.6 is OH. In
some embodiments of formula (III), G3 is unsubstituted. In other
embodiments, G3 is substituted. In various different embodiments,
G.sup.3 is linear C.sub.1-C.sub.24 alkylene or linear
C.sub.1-C.sub.24 alkenylene. In some other foregoing embodiments of
formula (III), R.sup.1 or R.sup.2, or both, is C.sub.6-C.sub.24
alkenyl. For example, in some embodiments, R.sup.1 and R.sup.2
each, independently have the following structure:
##STR00012##
[0620] wherein:
[0621] R.sup.7a and R.sup.7b are, at each occurrence, independently
H or C.sub.1-C.sub.12 alkyl; and a is an integer from 2 to 12,
wherein
[0622] R.sup.7a, R.sup.7b and a are each selected such that R.sup.1
and R.sup.2 each independently comprise from 6 to 20 carbon atoms.
For example, in some embodiments a is an integer ranging from 5 to
9 or from 8 to 12. In some of the foregoing embodiments of formula
(III), at least one occurrence of R.sup.7a is H. For example, in
some embodiments, R.sup.7a is H at each occurrence. In other
different embodiments of the foregoing, at least one occurrence of
R.sup.7b is C.sub.1-C.sub.8 alkyl. For example, in some
embodiments, C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl,
iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
[0623] In different embodiments of formula (III), R.sup.1 or
R.sup.2, or both, has one of the following structures:
##STR00013##
[0624] In preferred embodiments of the second aspect, the cationic
lipid of the LNP is a compound of formula III, wherein:
[0625] L.sup.1 and L.sup.2 are each independently --O(C.dbd.O)-- or
(C.dbd.O)--O--; and
[0626] R.sup.1 and R.sup.2 each independently have one of the
following structures:
##STR00014##
[0627] In some of the foregoing embodiments of formula (III),
R.sup.3 is OH, CN, --C(.dbd.O)OR.sup.4, --OC(.dbd.O)R.sup.4 or
--NHC(.dbd.O)R.sup.4. In some embodiments, R.sup.4 is methyl or
ethyl.
[0628] In preferred embodiments of the second aspect, the cationic
lipid of the LNP is a compound of formula II, wherein R.sup.3 is
OH.
[0629] In particularly preferred embodiment of the second aspect,
the artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
lipids thereby forming lipid nanoparticles (LNP), wherein the LNP
is selected from structures III-1 to III-36 (see Table 8).
TABLE-US-00013 TABLE 8 Representative lipid compounds derived from
formula (III) No. Structure III-1 ##STR00015## III-2 ##STR00016##
III-3 ##STR00017## III-4 ##STR00018## III-5 ##STR00019## III-6
##STR00020## III-7 ##STR00021## III-8 ##STR00022## III-9
##STR00023## III-10 ##STR00024## III-11 ##STR00025## III-12
##STR00026## III-13 ##STR00027## III-14 ##STR00028## III-15
##STR00029## III-16 ##STR00030## III-17 ##STR00031## III-18
##STR00032## III-19 ##STR00033## III-20 ##STR00034## III-21
##STR00035## III-22 ##STR00036## III-23 ##STR00037## III-24
##STR00038## III-25 ##STR00039## III-26 ##STR00040## III-27
##STR00041## III-28 ##STR00042## III-29 ##STR00043## III-30
##STR00044## III-31 ##STR00045## III-32 ##STR00046## III-33
##STR00047## III-34 ##STR00048## III-35 ##STR00049## III-36
##STR00050##
[0630] In some embodiments, the LNPs comprise a lipid of formula
(III), an artificial RNA of the first aspect, and, optionally, the
further artificial RNA of the second aspect, and one or more
excipient selected from neutral lipids, steroids and PEGylated
lipids. In some embodiments the lipid of formula (III) is compound
III-3. In some embodiments the lipid of formula (III) is compound
III-7.
[0631] In preferred embodiments, the LNP comprises a cationic lipid
selected from:
##STR00051##
[0632] In particularly preferred embodiment of the second aspect,
the artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
lipids thereby forming lipid nanoparticles (LNP), wherein the LNP
comprises the following cationic lipid (lipid according to formula
III-3 of Table 8):
##STR00052##
[0633] In certain embodiments, the cationic lipid as defined
herein, preferably as disclosed in Table 8, more preferably
cationic lipid compound III-3, is present in the LNP in an amount
from about 30 to about 95 mole percent, relative to the total lipid
content of the LNP. If more than one cationic lipid is incorporated
within the LNP, such percentages apply to the combined cationic
lipids.
[0634] In one embodiment, the cationic lipid is present in the LNP
in an amount from about 30 to about 70 mole percent. In one
embodiment, the cationic lipid is present in the LNP in an amount
from about 40 to about 60 mole percent, such as about 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59
or 60 mole percent, respectively. In embodiments, the cationic
lipid is present in the LNP in an amount from about 47 to about 48
mole percent, such as about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5,
47.6, 47.7, 47.8, 47.9, 50.0 mole percent, respectively, wherein
47.7 mole percent are particularly preferred.
[0635] In some embodiments, the cationic lipid is present in a
ratio of from about 20 mol % to about 70 or 75 mol % or from about
45 to about 65 mol % or about 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, or about 70 mol % of the total lipid present in the LNP. In
further embodiments, the LNPs comprise from about 25% to about 75%
on a molar basis of cationic lipid, e.g., from about 20 to about
70%, from about 35 to about 65%, from about 45 to about 65%, about
60%, about 57.5%, about 57.1%, about 50% or about 40% on a molar
basis (based upon 100% total moles of lipid in the lipid
nanoparticle). In some embodiments, the ratio of cationic lipid to
nucleic acid, preferably to the artificial RNA of the first aspect,
and, optionally, the further artificial RNA of the second aspect,
is from about 3 to about 15, such as from about 5 to about 13 or
from about 7 to about 11.
[0636] In some embodiments of the invention the LNP comprises a
combination or mixture of any the lipids described above.
[0637] Other suitable (cationic) lipids are disclosed in
WO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406,
WO2010/088537, WO2010/129709, WO2011/153493, US2011/0256175,
US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601,
WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620,
WO2017/099823, and WO2017/112865. In that context, the disclosures
of WO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406,
WO2010/088537, WO2010/129709, WO2011/153493, US2011/0256175,
US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601,
WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620,
WO2017/099823, and WO2017/112865 specifically relating to
(cationic) lipids suitable for LNPs are incorporated herewith by
reference.
[0638] In some embodiments, the lipid is selected from the group
consisting of 98N12-5, C12-200, and ckk-E12.
[0639] In some embodiments, amino or cationic lipids as defined
herein have at least one protonatable or deprotonatable group, such
that the lipid is positively charged at a pH at or below
physiological pH (e.g. pH 7.4), and neutral at a second pH,
preferably at or above physiological pH. It will, of course, be
understood that the addition or removal of protons as a function of
pH is an equilibrium process, and that the reference to a charged
or a neutral lipid refers to the nature of the predominant species
and does not require that all of lipids have to be present in the
charged or neutral form. Lipids having more than one protonatable
or deprotonatable group, or which are zwitterionic, are not
excluded and may likewise suitable in the context of the present
invention.
[0640] In some embodiments, the protonatable lipids have a pKa of
the protonatable group in the range of about 4 to about 11, e.g., a
pKa of about 5 to about 7.
[0641] LNPs can comprise two or more (different) cationic lipids.
The cationic lipids may be selected to contribute different
advantageous properties. For example, cationic lipids that differ
in properties such as amine pKa, chemical stability, half-life in
circulation, half-life in tissue, net accumulation in tissue, or
toxicity can be used in the LNP. In particular, the cationic lipids
can be chosen so that the properties of the mixed-LNP are more
desirable than the properties of a single-LNP of individual
lipids.
[0642] The amount of the permanently cationic lipid or lipidoid may
be selected taking the amount of the nucleic acid cargo into
account. In one embodiment, these amounts are selected such as to
result in an N/P ratio of the nanoparticle(s) or of the composition
in the range from about 0.1 to about 20. In this context, the N/P
ratio is defined as the mole ratio of the nitrogen atoms ("N") of
the basic nitrogen-containing groups of the lipid or lipidoid to
the phosphate groups ("P") of the RNA which is used as cargo. The
N/P ratio may be calculated on the basis that, for example, 1 .mu.g
RNA typically contains about 3 nmol phosphate residues, provided
that the RNA exhibits a statistical distribution of bases. The
"N"-value of the lipid or lipidoid may be calculated on the basis
of its molecular weight and the relative content of permanently
cationic and--if present--cationisable groups.
[0643] LNP in vivo characteristics and behavior can be modified by
addition of a hydrophilic polymer coating, e.g. polyethylene glycol
(PEG), to the LNP surface to confer steric stabilization.
Furthermore, LNPs can be used for specific targeting by attaching
ligands (e.g. antibodies, peptides, and carbohydrates) to its
surface or to the terminal end of the attached PEG chains (e.g. via
PEGylated lipids).
[0644] In some embodiments, the LNPs comprise a polymer conjugated
lipid. The term "polymer conjugated lipid" refers to a molecule
comprising both a lipid portion and a polymer portion. An example
of a polymer conjugated lipid is a PEGylated lipid. The term
"PEGylated lipid" refers to a molecule comprising both a lipid
portion and a polyethylene glycol portion. PEGylated lipids are
known in the art and include
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-s-DMG) and the like.
[0645] In certain embodiments, the LNP comprises an additional,
stabilizing-lipid which is a polyethylene glycol-lipid (PEGylated
lipid). Suitable polyethylene glycol-lipids include PEG-modified
phosphatidylethanolamine, PEG-modified phosphatidic acid,
PEG-modified ceramides (e.g. PEG-CerC14 or PEG-CerC20),
PEG-modified dialkylamines, PEG-modified diacylglycerols,
PEG-modified dialkylglycerols. Representative polyethylene
glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one
embodiment, the polyethylene glycol-lipid is N-[(methoxy
poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine
(PEG-c-DMA). In a preferred embodiment, the polyethylene
glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene
glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs
comprise a PEGylated diacylglycerol (PEG-DAG) such as
1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol
(PEG-DMG), a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG
succinate diacylglycerol (PEG-S-DAG) such as
4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)but-
anedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG
dialkoxypropylcarbamate such as
w-methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate
or
2,3-di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate.
[0646] In preferred embodiments of the second aspect, the
artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
lipids thereby forming lipid nanoparticles (LNP), wherein the LNP
additionally comprises a PEGylated lipid with the formula (IV):
##STR00053##
[0647] or a pharmaceutically acceptable salt, tautomer or
stereoisomer thereof, wherein R.sup.8 and R.sup.9 are each
independently a straight or branched, saturated or unsaturated
alkyl chain containing from 10 to 30 carbon atoms, wherein the
alkyl chain is optionally interrupted by one or more ester bonds;
and w has mean value ranging from 30 to 60.
[0648] In some of the foregoing embodiments of the PEGylated lipid
according to formula (IV), R.sup.8 and R.sup.9 are not both
n-octadecyl when w is 42. In some other embodiments, R.sup.8 and
R.sup.9 are each independently a straight or branched, saturated or
unsaturated alkyl chain containing from 10 to 18 carbon atoms. In
some embodiments, R.sup.8 and R.sup.9 are each independently a
straight or branched, saturated or unsaturated alkyl chain
containing from 12 to 16 carbon atoms. In some embodiments, R.sup.8
and R.sup.9 are each independently a straight or branched,
saturated or unsaturated alkyl chain containing 12 carbon atoms. In
some embodiments, R.sup.8 and R.sup.9 are each independently a
straight or branched, saturated or unsaturated alkyl chain
containing 14 carbon atoms. In other embodiments, R.sup.8 and
R.sup.9 are each independently a straight or branched, saturated or
unsaturated alkyl chain containing 16 carbon atoms. In still more
embodiments, R.sup.8 and R.sup.9 are each independently a straight
or branched, saturated or unsaturated alkyl chain containing 18
carbon atoms. In still other embodiments, R.sup.8 is a straight or
branched, saturated or unsaturated alkyl chain containing 12 carbon
atoms and R.sup.9 is a straight or branched, saturated or
unsaturated alkyl chain containing 14 carbon atoms.
[0649] In various embodiments, w spans a range that is selected
such that the PEG portion of the PEGylated lipid according to
formula (IV) has an average molecular weight of about 400 to about
6000 g/mol. In some embodiments, the average w is about 50.
[0650] In preferred embodiments of the second aspect, R.sup.8 and
R.sup.9 of the PEGylated lipid according to formula (IV) are
saturated alkyl chains.
[0651] In a particularly preferred embodiment of the second aspect,
the artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
lipids thereby forming lipid nanoparticles (LNP), wherein the LNP
additionally comprises a PEGylated lipid, wherein the PEG lipid is
of formula (IVa)
##STR00054##
[0652] wherein n has a mean value ranging from 30 to 60, such as
about 28 to about 32, about 30 to about 34, 32 to about 36, about
34 to about 38, 36 to about 40, about 38 to about 42, 40 to about
44, about 42 to about 46, 44 to about 48, about 46 to about 50, 48
to about 52, about 50 to about 54, 52 to about 56, about 54 to
about 58, 56 to about 60, about 58 to about 62. In preferred
embodiments, n is about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54. In
a most preferred embodiment n has a mean value of 49.
[0653] In other embodiments, the PEGylated lipid has one of the
following structures:
##STR00055##
[0654] wherein n is an integer selected such that the average
molecular weight of the PEGylated lipid is about 2500 g/mol, most
preferably n is about 49.
[0655] Further examples of PEG-lipids suitable in that context are
provided in US2015/0376115A1 and WO2015/199952, each of which is
incorporated by reference in its entirety.
[0656] In some embodiments, LNPs include less than about 3, 2, or 1
mole percent of PEG or PEG-modified lipid, based on the total moles
of lipid in the LNP. In further embodiments, LNPs comprise from
about 0.1% to about 20% of the PEG-modified lipid on a molar basis,
e.g., about 0.5 to about 10%, about 0.5 to about 5%. about 10%.
about 5%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%,
about 1%, about 0.5%, or about 0.3% on a molar basis (based on 100%
total moles of lipids in the LNP). In preferred embodiments, LNPs
comprise from about 1.0% to about 2.0% of the PEG-modified lipid on
a molar basis, e.g., about 1.2 to about 1.9%, about 1.2 to about
1.8%, about 1.3 to about 1.8%, about 1.4 to about 1.8%, about 1.5
to about 1.8%, about 1.6 to about 1.8%, in particular about 1.4%,
about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, most
preferably 1.7% (based on 100% total moles of lipids in the LNP).
In various embodiments, the molar ratio of the cationic lipid to
the PEGylated lipid ranges from about 100:1 to about 25:1.
[0657] In preferred embodiments, the LNP additionally comprises one
or more additional lipids which stabilize the formation of
particles during their formation (e.g. neutral lipid and/or one or
more steroid or steroid analogue).
[0658] In preferred embodiments of the second aspect, the
artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
lipids thereby forming lipid nanoparticles (LNP), wherein the LNP
additionally comprises one or more neutral lipid and/or one or more
steroid or steroid analogue.
[0659] Suitable stabilizing lipids include neutral lipids and
anionic lipids. The term "neutral lipid" refers to any one of a
number of lipid species that exist in either an uncharged or
neutral zwitterionic form at physiological pH. Representative
neutral lipids include diacylphosphatidylcholines,
diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro
sphingomyelins, cephalins, and cerebrosides.
[0660] In embodiments of the second aspect, the LNP additionally
comprises one or more neutral lipids, wherein the neutral lipid is
selected from the group comprising distearoylphosphatidylcholine
(DSPC), dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol
(DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and
1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
[0661] In some embodiments, the LNPs comprise a neutral lipid
selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various
embodiments, the molar ratio of the cationic lipid to the neutral
lipid ranges from about 2:1 to about 8:1.
[0662] In preferred embodiments of the second aspect, the neutral
lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The
molar ratio of the cationic lipid to DSPC may be in the range from
about 2:1 to 8:1.
[0663] In preferred embodiments of the second aspect, the steroid
is cholesterol. The molar ratio of the cationic lipid to
cholesterol may be in the range from about 2:1 to 1:1.
[0664] The sterol can be about 10 mol % to about 60 mol % or about
25 mol % to about 40 mol % of the lipid particle. In one
embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, or about 60 mol % of the total lipid present in the lipid
particle. In another embodiment, the LNPs include from about 5% to
about 50% on a molar basis of the sterol, e.g., about 15% to about
45%, about 20% to about 40%, about 48%, about 40%, about 38.5%,
about 35%, about 34.4%, about 31.5% or about 31% on a molar basis
(based upon 100% total moles of lipid in the lipid
nanoparticle).
[0665] Preferably, lipid nanoparticles (LNPs) comprise: (a) at
least one artificial RNA of the first aspect, and, optionally, the
further artificial RNA of the second aspect, (b) a cationic lipid,
(c) an aggregation reducing agent (such as polyethylene glycol
(PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic
lipid (such as a neutral lipid), and (e) optionally, a sterol.
[0666] In other preferred embodiments, lipid nanoparticles (LNPs)
comprise: (a) at least one artificial RNA encoding F of the first
aspect or a derivative or fragment thereof and at least one
artificial RNA encoding N, M, P, or M2-1 of the second aspect or a
derivative or fragment thereof, (b) a cationic lipid, (c) an
aggregation reducing agent (such as polyethylene glycol (PEG) lipid
or PEG-modified lipid), (d) optionally a non-cationic lipid (such
as a neutral lipid), and (e) optionally, a sterol.
[0667] In some embodiments, the LNPs comprise a lipid of formula
(III), an artificial RNA as defined herein, a neutral lipid, a
steroid and a PEGylated lipid. In preferred embodiments, the lipid
of formula (III) is lipid compound III-3, the neutral lipid is
DSPC, the steroid is cholesterol, and the PEGylated lipid is the
compound of formula (IVa).
[0668] In a preferred embodiment of the second aspect, the LNP
consists essentially of (i) at least one cationic lipid; (ii) a
neutral lipid; (iii) a sterol, e.g., cholesterol; and (iv) a
PEG-lipid, e.g. PEG-DMG or PEG-cDMA, in a molar ratio of about
20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15%
PEG-lipid.
[0669] In particularly preferred embodiments of the second aspect,
the artificial RNA of the first aspect and, optionally, the further
artificial RNA of the second aspect, is complexed with one or more
lipids thereby forming lipid nanoparticles (LNP), wherein the LNP
essentially consists of
(i) at least one cationic lipid as defined herein, preferably a
lipid of formula (III), more preferably lipid III-3; (ii) a neutral
lipid as defined herein, preferably
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) a steroid
or steroid analogue as defined herein, preferably cholesterol; and
(iv) a PEG-lipid as defined herein, e.g. PEG-DMG or PEG-cDMA,
preferably a PEGylated lipid of formula (IVa), wherein (i) to (iv)
are in a molar ratio of about 20-60% cationic lipid: 5-25% neutral
lipid: 25-55% sterol; 0.5-15% PEG-lipid.
[0670] In one preferred embodiment, the lipid nanoparticle
comprises: a cationic lipid with formula (III) and/or PEG lipid
with formula (IV), optionally a neutral lipid, preferably
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and optionally a
steroid, preferably cholesterol, wherein the molar ratio of the
cationic lipid to DSPC is optionally in the range from about 2:1 to
8:1, wherein the molar ratio of the cationic lipid to cholesterol
is optionally in the range from about 2:1 to 1:1.
[0671] In a particular preferred embodiment, the composition of the
second aspect comprising the artificial RNA of the first aspect
and, optionally, the further artificial RNA of the second aspect,
comprises lipid nanoparticles (LNPs), which have a molar ratio of
approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more
preferably 47.4:10:40.9:1.7 (i.e. proportion (mol %) of cationic
lipid (preferably lipid III-3), DSPC, cholesterol and PEG-lipid
((preferably PEG-lipid of formula (IVa) with n=49); solubilized in
ethanol).
[0672] In a particular preferred embodiment, the composition of the
second aspect comprises lipid nanoparticles (LNPs), which have a
molar ratio of approximately 50:10:38.5:1.5, preferably
47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e.
proportion (mol %) of cationic lipid (preferably lipid III-3),
DSPC, cholesterol and PEG-lipid ((preferably PEG-lipid of Formula
(IVa) with n=49); solubilized in ethanol), wherein the lipid
nanoparticles comprise at least one RNA encoding F (F0, F-del,
F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0,
F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2,
F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4,
F_DSCav1_mut5, F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6,
F_DSCav1_mut7, F-del_DSCav1_mut7, F_DSCav1_mut8, or
F-del_DSCav1_mut8), and, additionally lipid nanoparticles (LNPs),
which have a molar ratio of approximately 50:10:38.5:1.5,
preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7
(i.e. proportion (mol %) of cationic lipid (preferably lipid
III-3), DSPC, cholesterol and PEG-lipid ((preferably PEG-lipid of
Formula (IVa) with n=49); solubilized in ethanol), wherein the
lipid nanoparticles comprise at least one RNA encoding M, N, M2-1,
or P.
[0673] In a particular preferred embodiment, the composition of the
second aspect comprises lipid nanoparticles (LNPs), which have a
molar ratio of approximately 50:10:38.5:1.5, preferably
47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e.
proportion (mol %) of cationic lipid (preferably lipid III-3),
DSPC, cholesterol and PEG-lipid ((preferably PEG-lipid of Formula
(IVa) with n=49); solubilized in ethanol), wherein the lipid
nanoparticles comprise at least one RNA encoding F (F0, F-del,
F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0,
F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2,
F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4,
F_DSCav1_mut5, F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6,
F_DSCav1_mut7, F-del_DSCav1_mut7, F_DSCav1_mut8, or
F-del_DSCav1_mut8), and at least one RNA encoding M, N, M2-1, or
P.
[0674] The total amount of RNA in the lipid nanoparticles may vary
and is defined depending on the e.g. RNA to total lipid w/w ratio.
In one embodiment of the invention the artificial RNA to total
lipid ratio is less than 0.06 w/w, preferably between 0.03 w/w and
0.04 w/w.
[0675] In various embodiments, the LNP as defined herein have a
mean diameter of from about 50 nm to about 200 nm, from about 60 nm
to about 200 nm, from about 70 nm to about 200 nm, from about 80 nm
to about 200 nm, from about 90 nm to about 200 nm, from about 90 nm
to about 190 nm, from about 90 nm to about 180 nm, from about 90 nm
to about 170 nm, from about 90 nm to about 160 nm, from about 90 nm
to about 150 nm, from about 90 nm to about 140 nm, from about 90 nm
to about 130 nm, from about 90 nm to about 120 nm, from about 90 nm
to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm
to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35
nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm,
85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125
nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm,
190 nm, or 200 nm and are substantially non-toxic. As used herein,
the mean diameter may be represented by the z-average as determined
by dynamic light scattering as commonly known in the art.
[0676] In another preferred embodiment of the invention the lipid
nanoparticles have a hydrodynamic diameter in the range from about
50 nm to about 300 nm, or from about 60 nm to about 250 nm, from
about 60 nm to about 150 nm, or from about 60 nm to about 120 nm,
respectively.
[0677] According to further embodiments, the composition of the
second aspect may comprise at least one adjuvant. Suitably, the
adjuvant is preferably added to enhance the immunostimulatory
properties of the composition.
[0678] The term "adjuvant" as used herein will be recognized and
understood by the person of ordinary skill in the art, and is for
example intended to refer to a pharmacological and/or immunological
agent that may modify, e.g. enhance, the effect of other agents
(herein: the effect of the artificial nucleic acid of the
invention) or that may be suitable to support administration and
delivery of the composition. The term "adjuvant" refers to a broad
spectrum of substances. Typically, these substances are able to
increase the immunogenicity of antigens. For example, adjuvants may
be recognized by the innate immune systems and, e.g., may elicit an
innate immune response (that is, a non-specific immune response).
"Adjuvants" typically do not elicit an adaptive immune response. In
the context of the invention, adjuvants may enhance the effect of
the antigenic peptide or protein provided by the artificial nucleic
acid as defined herein or the polyprotein as defined herein.
[0679] In that context, the at least one adjuvant may be selected
from any adjuvant known to a skilled person and suitable for the
present case, i.e. supporting the induction of an immune response
in a subject, e.g. in a human subject.
[0680] Accordingly, the composition of the second aspect may
comprise at least one adjuvant, wherein the at least one adjuvant
may be suitably selected from any adjuvant provided in of published
PCT application WO2016/203025.
[0681] Adjuvants disclosed in any of the claims 2 to 17 of
WO2016/203025, preferably adjuvants disclosed in claim 17 of
WO2016/203025 are particularly suitable, the specific content
relating thereto herewith incorporated by reference.
[0682] The composition of the second aspect may comprise, besides
the components specified herein, at least one further component
which may be selected from the group consisting of further antigens
(e.g. in the form of a peptide or protein) or further
antigen-encoding nucleic acids; a further immunotherapeutic agent;
one or more auxiliary substances (cytokines, such as monokines,
lymphokines, interleukins or chemokines); or any further compound,
which is known to be immune stimulating due to its binding affinity
(as ligands) to human Toll-like receptors; and/or an adjuvant
nucleic acid, preferably an immunostimulatory RNA (isRNA), e.g.
CpG-RNA etc.
[0683] Polypeptide and Composition Comprising the Polypeptide
[0684] In a third aspect, the present invention provides a
polypeptide, preferably an antigenic polypeptide, wherein the
polypeptide comprises an amino acid sequence or a fragment thereof
encoded by the artificial RNA of the first aspect.
[0685] In embodiments, the polypeptide has an amino acid sequence
which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to
SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481,
3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540,
7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047,
15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630,
17999, 18368, 18737, 19106, 19475 or a variant of any of these
polypeptides.
[0686] In preferred embodiments, the polypeptide has an amino acid
sequence which is identical or at least 70%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical to SEQ ID NOs: 1267, 2005, 2743, 3481, 4219, 4957, 5695,
6433, 7171, 7909, 12833, 14309, 15047, 15785, 13571, 16523, 17261,
17999, 18737, 19475 or a variant of any of these polypeptides.
[0687] In a fourth aspect, the invention relates to an immunogenic
composition, comprising the polypeptide of the third aspect. In
preferred embodiments, the composition of the fourth aspect may
additionally comprise at least one pharmaceutically acceptable
carrier as defined herein. In preferred embodiments, the
composition of the fourth aspect may additionally comprise at least
one artificial RNA of the first aspect and, optionally, at least
one further artificial RNA of the second aspect, or an RNA
composition of the second aspect.
[0688] Notably, embodiments relating to the composition of the
second aspect or the vaccine of the fifth aspect may likewise be
read on and be understood as suitable embodiments of the
composition of the fourth aspect.
[0689] Vaccine:
[0690] In a fifth aspect, the present invention provides a vaccine
wherein the vaccine comprises the RNA of the first aspect, and,
optionally, the composition of the second aspect, the polypeptide
of the third aspect, or the composition of the fourth aspect.
[0691] Notably, embodiments relating to the composition of the
second aspect or the composition of the fourth aspect may likewise
be read on and be understood as suitable embodiments of the vaccine
of the fifth aspect. Also, embodiments relating to the vaccine of
the fifth aspect may likewise be read on and be understood as
suitable embodiments of the composition of the second aspect
(comprising the RNA of the first aspect and, optionally, the
further artificial RNA of the second aspect) or the composition of
the fourth aspect (comprising the polypeptide of the third
aspect).
[0692] The term "vaccine" will be recognized and understood by the
person of ordinary skill in the art, and is for example intended to
be a prophylactic or therapeutic material providing at least one
epitope or antigen, preferably an immunogen. In the context of the
invention the antigen or antigenic function is provided by the
inventive RNA of the first aspect (said RNA comprising a coding
sequence encoding a antigenic peptide or protein derived from RSV F
protein), the composition of the second aspect (comprising the RNA
of the first aspect and, optionally, the further artificial RNA of
the second aspect), the polypeptide of the third aspect, or the
composition of the fourth aspect (comprising said polypeptide).
[0693] In preferred embodiments of the fifth aspect, the vaccine
comprises the RNA of the first aspect, the composition of the
second aspect (comprising the RNA of the first aspect and,
optionally, the further artificial RNA of the second aspect), the
polypeptide of the third aspect, or the composition of the fourth
aspect wherein said RNA, said composition of the second aspect,
said polypeptide, or said composition of the fourth aspect
(comprising said polypeptide) elicits an adaptive immune
response.
[0694] In particularly preferred embodiments, the vaccine comprises
the RNA of the first aspect or the composition of the second aspect
wherein said RNA or said composition elicits an adaptive immune
response, preferably an adaptive immune response against RSV.
[0695] In particularly preferred embodiments, the vaccine comprises
the RNA of the first aspect or the composition of the second aspect
wherein said RNA or said composition induces a T cell immune
response, According to a preferred embodiment of the fifth aspect,
the vaccine as defined herein may further comprise a
pharmaceutically acceptable carrier and optionally at least one
adjuvant as specified in the context of the second aspect.
[0696] Suitable adjuvants in that context may be selected from
adjuvants disclosed in claim 17 of WO2016/203025.
[0697] In a preferred embodiment, the vaccine is a monovalent
vaccine.
[0698] In embodiments the vaccine is a polyvalent vaccine
comprising a plurality or at least more than one of the artificial
RNAs as defined in the context of the first aspect, and,
optionally, at least more than one of the further artificial RNAs
as defined in the context of the second aspect. Embodiments
relating to a polyvalent composition as disclosed in the context of
the second aspect may likewise be read on and be understood as
suitable embodiments of the polyvalent vaccine of the fifth
aspect.
[0699] The vaccine of the fifth aspect typically comprises a safe
and effective amount of the RNA of the first aspect and,
optionally, the further RNA of the second aspect. As used herein,
"safe and effective amount" means an amount of the RNA that is
sufficient to significantly induce a positive modification of a
disease or disorder related to an infection with a RSV. At the same
time, a "safe and effective amount" is small enough to avoid
serious side-effects. In relation to the vaccine or composition of
the present invention, the expression "safe and effective amount"
preferably means an amount of the RNA that is suitable for
stimulating the adaptive immune system in such a manner that no
excessive or damaging immune reactions are achieved but,
preferably, also no such immune reactions below a measurable
level.
[0700] A "safe and effective amount" of the RNA of the composition
or vaccine as defined above will furthermore vary in connection
with the particular condition to be treated and also with the age
and physical condition of the patient to be treated, the severity
of the condition, the duration of the treatment, the nature of the
accompanying therapy, of the particular pharmaceutically acceptable
carrier used, and similar factors, within the knowledge and
experience of the accompanying medical doctor. Moreover, the "safe
and effective amount" of the RNA, the composition, the vaccine may
depend from application route (intradermal, intramuscular),
application device (jet injection, needle injection, microneedle
patch) and/or complexation (protamine complexation or LNP
encapsulation). Moreover, the "safe and effective amount" of the
artificial RNA, the composition, the vaccine may depend from the
condition of the treated subject (infant, pregnant women,
immunocompromised human subject etc.). Accordingly, the suitable
"safe and effective amount" has to be adapted accordingly and will
be chosen and defined by the skilled person.
[0701] The vaccine can be used according to the invention for human
medical purposes and also for veterinary medical purposes (mammals,
vertebrates, avian species), as a pharmaceutical composition, or as
a vaccine.
[0702] In a preferred embodiment, the RNA, the composition
(comprising said RNA and, optionally, a further RNA), the
polypeptide, the composition (comprising said polypeptide), or the
vaccine is provided in lyophilized form (using e.g. lyophilisation
methods as described in WO2016/165831, WO2011/069586, WO2016/184575
or WO2016/184576). Preferably, the lyophilized RNA, the lyophilized
composition, the lyophilized polypeptide, the lyophilized
composition comprising the polypeptide, or the lyophilized vaccine
is reconstituted in a suitable buffer, advantageously based on an
aqueous carrier, prior to administration, e.g. Ringer-Lactate
solution or a phosphate buffer solution.
[0703] Accordingly, the pharmaceutically acceptable carrier as used
herein preferably includes the liquid or non-liquid basis of the
inventive vaccine. If the inventive vaccine is provided in liquid
form, the carrier will be water, typically pyrogen-free water;
isotonic saline or buffered (aqueous) solutions, e.g. phosphate,
citrate etc. buffered solutions. Preferably, Ringer-Lactate
solution is used as a liquid basis for the vaccine or the
composition according to the invention as described in
WO2006/122828, the disclosure relating to suitable buffered
solutions incorporated herewith by reference.
[0704] The choice of a pharmaceutically acceptable carrier as
defined herein is determined, in principle, by the manner, in which
the pharmaceutical composition(s) or vaccine according to the
invention is administered. The vaccine can be administered, for
example, systemically or locally. Routes for systemic
administration in general include, for example, transdermal, oral,
parenteral routes, including subcutaneous, intravenous,
intramuscular, intra-arterial, intradermal and intraperitoneal
injections and/or intranasal administration routes. Routes for
local administration in general include, for example, topical
administration routes but also intradermal, transdermal,
subcutaneous, or intramuscular injections or intralesional,
intracranial, intrapulmonal, intracardial, intraarticular and
sublingual injections. More preferably, composition or vaccines
according to the present invention may be administered by an
intradermal, subcutaneous, or intramuscular route, preferably by
injection, which may be needle-free and/or needle injection.
Compositions/vaccines are therefore preferably formulated in liquid
or solid form. The suitable amount of the vaccine or composition
according to the invention to be administered can be determined by
routine experiments, e.g. by using animal models. Such models
include, without implying any limitation, rabbit, sheep, mouse,
rat, dog and non-human primate models. Preferred unit dose forms
for injection include sterile solutions of water, physiological
saline or mixtures thereof. The pH of such solutions should be
adjusted to about 7.4. Suitable carriers for injection include
hydrogels, devices for controlled or delayed release, polylactic
acid and collagen matrices. Suitable pharmaceutically acceptable
carriers for topical application include those which are suitable
for use in lotions, creams, gels and the like. If the inventive
compositionor vaccine is to be administered perorally, tablets,
capsules and the like are the preferred unit dose form. The
pharmaceutically acceptable carriers for the preparation of unit
dose forms which can be used for oral administration are well known
in the prior art. The choice thereof will depend on secondary
considerations such as taste, costs and storability, which are not
critical for the purposes of the present invention, and can be made
without difficulty by a person skilled in the art.
[0705] The inventive vaccine or composition as defined herein can
additionally contain one or more auxiliary substances as defined
above in order to further increase the immunogenicity. A
synergistic action of the nucleic acid contained in the inventive
composition and of an auxiliary substance, which may be optionally
be co-formulated (or separately formulated) with the inventive
vaccine or composition as described above, is preferably achieved
thereby. Such immunogenicity increasing agents or compounds may be
provided separately (not co-formulated with the inventive vaccine
or composition) and administered individually.
[0706] Further additives which may be included in the inventive
vaccine or composition are emulsifiers, such as for example, Tween;
wetting agents, such as, for example, sodium lauryl sulfate;
colouring agents; taste-imparting agents, pharmaceutical carriers;
tablet-forming agents; stabilizers; antioxidants;
preservatives.
[0707] Kit or Kit of Parts, Application, Medical Uses, Method of
Treatment:
[0708] In a sixth aspect, the present invention provides a kit or
kit of parts, wherein the kit or kit of parts comprises the RNA of
the first aspect, the composition of the second aspect (comprising
said RNA), the polypeptide of the third aspect, the composition of
the fourth aspect (comprising said polypeptide), and/or the vaccine
of the fifth aspect, optionally comprising a liquid vehicle for
solubilising, and optionally technical instructions providing
information on administration and dosage of the components.
[0709] In a preferred embodiment, the kit of the sixth aspect
comprises at least the following components [0710] a) at least one
RNA of the first aspect, preferably encoding at least one antigenic
peptide or protein derived from a RSV fusion (F) protein (RNA
sequences preferably selected from Table 5 or 6), wherein said
artificial RNA is preferably complexed with one or more lipids
thereby forming lipid nanoparticles (LNP); and [0711] b) at least
one, two, or three further artificial RNA encoding an antigenic
peptide or protein derived from RSV selected from M, N, M2-1, or P
(RNA sequences preferably selected from Table 7A), wherein said
further RNA is preferably complexed with one or more lipids thereby
forming lipid nanoparticles (LNP), wherein components a) and b) are
provided as separate entities or as a single entity.
[0712] In a preferred embodiment, the kit of the sixth aspect
comprises at least the following components [0713] a) at least one
RNA of the first aspect, preferably encoding at least one antigenic
peptide or protein derived from a RSV fusion (F) protein (RNA
sequences preferably selected from Table 5 or 6), wherein said
artificial RNA is preferably complexed with one or more lipids
thereby forming lipid nanoparticles (LNP); and [0714] b) at least
one further artificial RNA encoding an antigenic peptide or protein
derived from RSV selected from M (RNA sequences preferably selected
from Table 7A, Column A), wherein said further RNA is preferably
complexed with one or more lipids thereby forming lipid
nanoparticles (LNP); and [0715] c) at least one further artificial
RNA encoding an antigenic peptide or protein derived from RSV
selected from P (RNA sequences preferably selected from Table 7A,
Column C), wherein said further RNA is preferably complexed with
one or more lipids thereby forming lipid nanoparticles (LNP),
[0716] wherein components a), b) and c) are provided as separate
entities or as a single entity.
[0717] In a preferred embodiment, the kit of the sixth aspect
comprises at least the following components [0718] a) at least one
RNA of the first aspect, preferably encoding at least one antigenic
peptide or protein derived from a RSV fusion (F) protein (RNA
sequences preferably selected from Table 5 or 6), wherein said
artificial RNA is preferably complexed with one or more lipids
thereby forming lipid nanoparticles (LNP); and [0719] b) at least
one further artificial RNA encoding an antigenic peptide or protein
derived from RSV selected from M (RNA sequences preferably selected
from Table 7A, Column A), wherein said further RNA is preferably
complexed with one or more lipids thereby forming lipid
nanoparticles (LNP); and [0720] c) at least one further artificial
RNA encoding an antigenic peptide or protein derived from RSV
selected from P (RNA sequences preferably selected from Table 7A,
Column C), wherein said further RNA is preferably complexed with
one or more lipids thereby forming lipid nanoparticles (LNP); and
[0721] d) at least one further artificial RNA encoding an antigenic
peptide or protein derived from RSV selected from P (RNA sequences
preferably selected from Table 7A, Column B), wherein said further
RNA is preferably complexed with one or more lipids thereby forming
lipid nanoparticles (LNP),
[0722] wherein components a), b), c) and d) are provided as
separate entities or as a single entity.
[0723] The kit may further comprise additional components as
described in the context of the composition of the second aspect,
the polypeptide of the third aspect, the composition of the fourth
aspect, and/or the vaccine of the fifth aspect.
[0724] The technical instructions of said kit may contain
information about administration and dosage and patient groups.
Such kits, preferably kits of parts, may be applied e.g. for any of
the applications or uses mentioned herein, preferably for the use
of the RNA of the first aspect, the composition of the second
aspect, the polypeptide of the third aspect, the composition of the
fourth aspect, or the vaccine of the fifth aspect, for the
treatment or prophylaxis of an infection or diseases caused by RSV
or disorders related thereto. Preferably, the RNA of the first
aspect, the composition of the second aspect, the polypeptide of
the third aspect, the composition of the fourth aspect, or the
vaccine of the fifth aspect is provided in a separate part of the
kit, wherein the RNA of the first aspect, the composition of the
second aspect, the polypeptide of the third aspect, the composition
of the fourth aspect, or the vaccine of the fifth aspect is
preferably lyophilised. The kit may further contain as a part a
vehicle (e.g. buffer solution) for solubilising the RNA of the
first aspect, the composition of the second aspect, the polypeptide
of the third aspect, the composition fourth aspect, or the vaccine
of the fifth aspect.
[0725] In preferred embodiments, the kit or kit of parts as defined
herein comprises Ringer lactate solution.
[0726] Any of the above kits may be used in a treatment or
prophylaxis as defined herein. More preferably, any of the above
kits may be used as a vaccine, preferably a vaccine against
infections caused by RSV as defined herein.
[0727] Medical Use:
[0728] In a further aspect, the present invention relates to the
first medical use of the RNA of the first aspect, the composition
of the second aspect, the polypeptide of the third aspect, the
composition of the fourth aspect, the vaccine of the fifth aspect,
or the kit or kit of parts of the sixth aspect.
[0729] Accordingly, the RNA of the first aspect, the composition of
the second aspect, the polypeptide of the third aspect, the
composition of the fourth aspect, the vaccine of the fifth aspect,
or the kit or kit of parts of the sixth aspect is for use as a
medicament.
[0730] The present invention furthermore provides several
applications and uses of the RNA of the first aspect, the
composition of the second aspect, the polypeptide of the third
aspect, the composition of the fourth aspect, the vaccine of the
fifth aspect, or the kit or kit of parts of the sixth aspect.
[0731] In particular, said RNA, composition (comprising said RNA),
polypeptide, composition (comprising said polypeptide), vaccine, or
the kit or kit of parts may be used for human medical purposes and
also for veterinary medical purposes, preferably for human medical
purposes.
[0732] In particular, said RNA, composition (comprising said RNA),
polypeptide, composition (comprising said polypeptide), vaccine, or
the kit or kit of parts may is for use as a medicament for human
medical purposes, wherein said RNA, composition (comprising said
RNA), polypeptide, composition (comprising said polypeptide),
vaccine, or the kit or kit of parts may be particularly suitable
for young infants, newborns, immunocompromised recipients, as well
as pregnant and breast-feeding women and elderly people.
[0733] In yet another aspect, the present invention relates to the
second medical use of the RNA of the first aspect, the composition
of the second aspect, the polypeptide of the third aspect, the
composition of the fourth aspect, the vaccine of the fifth aspect,
or the kit or kit of parts of the sixth aspect.
[0734] Accordingly, the RNA of the first aspect, the composition of
the second aspect, the polypeptide of the third aspect, the
composition of the fourth aspect, the vaccine of the fifth aspect,
or the kit or kit of parts of the sixth aspect is for use in the
treatment or prophylaxis of an infection with a pathogen (e.g. a
virus), in particular with Respiratory syncytial virus (RSV), or a
disorder related to such an infection.
[0735] In particular, the RNA of the first aspect, the composition
of the second aspect, the polypeptide of the third aspect, the
composition of the fourth aspect, the vaccine of the fifth aspect,
or the kit or kit of parts of the sixth aspect may be used in the
treatment or prophylaxis of an infection with a virus, in
particular with RSV, or a disorder related to such an infection for
human and also for veterinary medical purposes, preferably for
human medical purposes.
[0736] In particular, said RNA, composition (comprising said RNA),
polypeptide, composition (comprising said polypeptide), vaccine, or
the kit or kit of parts may be particularly suitable for young
infants, newborns, immunocompromised recipients, as well as
pregnant and breast-feeding women and elderly people for use in the
treatment or prophylaxis of an infection with RSV.
[0737] As used herein, "a disorder related to a RSV infection" may
preferably comprise a typical symptom or a complication of an RSV
infection.
[0738] Particularly, the artificial RNA of the first aspect, the
composition of the second aspect, the polypeptide of the third
aspect, the composition of the fourth aspect, the vaccine of the
fifth aspect, or the kit or kit of parts of the sixth aspect may be
used in a method of prophylactic (pre-exposure prophylaxis or
post-exposure prophylaxis) and/or therapeutic treatment of
infections caused by RSV.
[0739] The composition or the vaccine as defined herein may
preferably be administered locally. In particular, composition or
vaccines may be administered by an intradermal, subcutaneous,
intranasal, or intramuscular route. Inventive compositions or
vaccines of the invention are therefore preferably formulated in
liquid (or sometimes in solid) form. In embodiments, the inventive
vaccine may be administered by conventional needle injection or
needle-free jet injection. Preferred in that context is the RNA,
the composition, the vaccine is administered by intramuscular
needle injection.
[0740] The term "jet injection", as used herein, refers to a
needle-free injection method, wherein a fluid (vaccine, composition
of the invention) containing e.g. at least one RNA of the first
aspect is forced through an orifice, thus generating an ultra-fine
liquid stream of high pressure that is capable of penetrating
mammalian skin and, depending on the injection settings,
subcutaneous tissue or muscle tissue. In principle, the liquid
stream perforates the skin, through which the liquid stream is
pushed into the target tissue. Preferably, jet injection is used
for intradermal, subcutaneous or intramuscular injection of the
RNA, the compositions, the vaccines disclosed herein.
[0741] In embodiments, the RNA as comprised in a composition or
vaccine as defined herein is provided in an amount of about 100 ng
to about 500 .mu.g, in an amount of about 1 .mu.g to about 200
.mu.g, in an amount of about 1 .mu.g to about 100 .mu.g, in an
amount of about 5 .mu.g to about 100 .mu.g, preferably in an amount
of about 10 .mu.g to about 50 .mu.g, specifically, in an amount of
about 5 .mu.g, 10 .mu.g, 15 .mu.g, 20 .mu.g, 25 .mu.g, 30 .mu.g, 35
.mu.g, 40 .mu.g, 45 .mu.g, 50 .mu.g, 55 .mu.g, 60 .mu.g, 65 .mu.g,
70 .mu.g, 75 .mu.g, 80 .mu.g, 85 .mu.g, 90 .mu.g, 95 .mu.g or 100
.mu.g.
[0742] Depending from application route (intradermal,
intramuscular, intranasal), application device (jet injection,
needle injection, microneedle patch) and/or complexation
(preferably LNP encapsulation) the suitable amount has to be
adapted accordingly and will be chosen and defined by the skilled
person.
[0743] The immunization protocol for the treatment or prophylaxis
of an infection as defined herein, i.e. the immunization of a
subject against a RSV, typically comprises a series of single doses
or dosages of the composition or the vaccine. A single dosage, as
used herein, refers to the initial/first dose, a second dose or any
further doses, respectively, which are preferably administered in
order to "boost" the immune reaction.
[0744] In one embodiment, the immunization protocol for the
treatment or prophylaxis of an infection as defined herein, i.e.
the immunization of a subject against a RSV, comprises one single
doses of the composition or the vaccine.
[0745] In preferred embodiments, the immunization protocol for the
prophylaxis of an infection with RSV comprises at least one single
dose of the composition or the vaccine as defined herein, wherein
said at least one single dose is administered to a pregnant women
for maternal immunization, thereby achieving immunization of the
unborn child and/or wherein said at least one single dose is
administered to a breast-feeding women for thereby achieving
passive immunization of the breast-fed child.
[0746] The treatment or prophylaxis as defined above may comprise
the administration of a further active pharmaceutical ingredient.
In the case of the inventive vaccine or composition, which is based
on the artificial RNA of the first aspect, a polypeptide,
preferably of the third aspect may be co-administered as a further
active pharmaceutical ingredient.
[0747] For example, at least one RSV protein or peptide as
described herein, or a fragment or variant thereof, may be
co-administered in order to induce or enhance an immune response.
Further, two distinct artificial RNAs of the first aspect and,
optionally further RNAs of the second aspect may be administered at
different time points, preferably in a prime-boost scenario, e.g.
using a composition comprising at least one RSV polypeptide as
prime vaccination and a composition/vaccine comprising at least one
artificial RNA of the first aspect as boost vaccination.
[0748] Suitably, the treatment or prophylaxis as defined above
comprises the administration of a further active pharmaceutical
ingredient, wherein the further active pharmaceutical ingredient
may be an immunotherapeutic agent that can be selected from
immunoglobulins, preferably IgGs, monoclonal or polyclonal
antibodies, polyclonal serum or sera, etc., most preferably
immunoglobulins directed against a RSV protein or peptide as
defined herein. Preferably, such a further immunotherapeutic agent
may be provided as a peptide/protein or may be encoded by a nucleic
acid, preferably by a DNA or an RNA, more preferably an mRNA. Such
an immunotherapeutic agent allows providing passive vaccination
additional to active vaccination triggered by the inventive
artificial RNA or by the inventive polypeptide.
[0749] Method of Treatment and Use, Diagnostic Method and Use:
[0750] In another aspect, the present invention relates to a method
of treating or preventing a disorder.
[0751] In preferred embodiments, the present invention relates to a
method of treating or preventing a disorder, wherein the method
comprises applying or administering to a subject in need thereof
the RNA of the first aspect, the composition of the second aspect,
the polypeptide of the third aspect, the composition of the fourth
aspect, the vaccine of the fifth aspect, or the kit or kit of parts
of the sixth aspect.
[0752] In preferred embodiments, the disorder is an infection with
Respiratory syncytial virus (RSV), or a disorder related to such an
infection.
[0753] In preferred embodiments, the present invention relates to a
method of treating or preventing a disorder, wherein the method
comprises applying or administering to a subject in need thereof
the RNA of the first aspect, the composition of the second aspect,
the polypeptide of the third aspect, the composition of the fourth
aspect, the vaccine of the fifth aspect, or the kit or kit of parts
of the sixth aspect, wherein the subject in need is preferably a
mammalian subject. In particularly preferred embodiments, the
mammalian subject is a human subject, particularly an infant, a
newborn, a pregnant women, a breast-feeding woman, an elderly, or
an immunocompromised human subject.
[0754] In particular, such a method may preferably comprise the
steps of: [0755] a) providing the RNA of the first aspect, the
composition of the second aspect, the polypeptide of the third
aspect, the composition of the fourth aspect, the vaccine of the
fifth aspect, or the kit or kit of parts of the sixth aspect;
[0756] b) applying or administering said RNA of the first aspect,
composition of the second aspect, polypeptide of the third aspect,
composition of the fourth aspect, vaccine of the fifth aspect, or
kit or kit of parts of the sixth aspect to a tissue or an organism;
[0757] c) optionally, administering immunoglobulin (IgGs) against a
RSV; [0758] d) optionally, administering a further substance
(adjuvant, auxiliary substance, further antigen).
[0759] According to a further aspect, the present invention also
provides a method for expression of at least one polypeptide
comprising at least one peptide or protein derived from a RSV, or a
fragment or variant thereof, wherein the method preferably
comprises the following steps: [0760] a) providing the RNA of the
first aspect or the composition of the second aspect; and [0761] b)
applying or administering said RNA or composition to an expression
system (cells), a tissue, an organism.
[0762] The method may be applied for laboratory, for research, for
diagnostic, for commercial production of peptides or proteins
and/or for therapeutic purposes. The method may furthermore be
carried out in the context of the treatment of a specific disease,
particularly in the treatment of infectious diseases, particularly
RSV infections.
[0763] Likewise, according to another aspect, the present invention
also provides the use of the artificial RNA of the first aspect,
the composition of the second aspect, the polypeptide of the third
aspect, the composition of the fourth aspect, the vaccine of the
fifth aspect, or the kit or kit of parts of the sixth aspect
preferably for diagnostic or therapeutic purposes, e.g. for
expression of an encoded RSV antigenic peptide or protein, e.g. by
applying or administering said RNA, composition comprising said
RNA, vaccine comprising said RNA, e.g. to a cell-free expression
system, a cell (e.g. an expression host cell or a somatic cell), a
tissue or an organism. In specific embodiments, applying or
administering said RNA, composition comprising said RNA, vaccine
comprising said RNA to a tissue or an organism is followed by e.g.
a step of obtaining induced RSV F antibodies e.g. RSV F specific
(monoclonal) antibodies.
[0764] The use may be applied for a (diagnostic) laboratory, for
research, for diagnostics, for commercial production of peptides,
proteins, or RSV antibodies and/or for therapeutic purposes. The
use may be carried out in vitro, in vivo or ex vivo. The use may
furthermore be carried out in the context of the treatment of a
specific disease, particularly in the treatment of an RSV infection
or a related disorder.
[0765] In a particularly preferred embodiment, the invention
provides the RNA of the first aspect, the composition of the second
aspect, the polypeptide of the third aspect, the composition of the
fourth aspect, the vaccine of the fifth aspect, or the kit or kit
of parts of the sixth aspect for use as a medicament, for use in
treatment or prophylaxis, preferably treatment or prophylaxis of an
RSV infection or a related disorder, or for use as a vaccine.
BRIEF DESCRIPTION OF LISTS AND TABLES
[0766] List 1: Suitable RSV virus strains
[0767] List 2: NCBI Protein Accession numbers of suitable RSV
fusion (F) proteins
[0768] Table 1: Preferred RSV F protein antigen designs
[0769] Table 2: Human codon usage table with frequencies indicated
for each amino acid
[0770] Table 3A: Preferred coding sequences encoding RSV F (columns
A-J), derived from HRSV(A2)
[0771] Table 3B: Preferred coding sequences encoding RSV F (columns
A-J), derived from HRSV(Memphis-37)
[0772] Table 4A: Preferred coding sequences encoding RSV F (columns
K-V), derived from HRSV(A2)
[0773] Table 4B: Preferred coding sequences encoding RSV F (columns
K-V), derived from HRSV(Memphis-37)
[0774] Table 5A: Preferred mRNA constructs encoding RSV F (columns
A-J), derived from HRSV(A2)
[0775] Table 5B: Preferred mRNA constructs encoding RSV F (columns
A-J), derived from HRSV(Memphis-37)
[0776] Table 6A: Preferred mRNA constructs encoding RSV F (column
K-V), derived from HRSV(A2)
[0777] Table 6B: Preferred mRNA constructs encoding RSV F (column
K-V), derived from HRSV(Memphis-37)
[0778] Table 7A: Preferred further coding sequences and mRNA
constructs of the composition or vaccine
[0779] Table 7B: Suitable combinations of RNA constructs
[0780] Table 8: Representative lipid compounds derived from formula
(III)
[0781] Table 9: mRNA constructs used in the present examples (see
Examples section)
[0782] Table 10: Animal groups and vaccination schedule of Example
2 (see Examples section)
[0783] Table 11: Animal groups and vaccination schedule of Example
3 (see Examples section)
[0784] Table 12: mRNA constructs with different UTR combinations of
Example 4 (see Examples section)
[0785] Table 13: Animal groups and vaccination schedule of Example
5 (see Examples section)
[0786] Table 14: Animal groups and vaccination schedule of Example
6 (see Examples section)
[0787] Table 15: Animal groups and vaccination schedule of Example
7 (see Examples section)
[0788] Table 15: Animal groups and vaccination schedule of Example
7 (see Examples section)
[0789] Table 16: Overview of mRNA constructs used in Example 8 (see
Examples section)
[0790] Table 17: Animal groups and vaccination schedule of Example
9 (see Examples section)
[0791] Table 18: Animal groups and vaccination schedule of Example
10 (see Examples section)
[0792] Table 19: Overview of mRNA constructs used in Example 11
(see Examples section)
[0793] Table 20: Animal groups and vaccination schedule of Example
12 (see Examples section)
BRIEF DESCRIPTION OF THE DRAWINGS
[0794] FIG. 1 shows that LNP-formulated mRNA encoding RSV F-protein
(F-del_DSCav1) induces high virus neutralization titers (VNTs) in
serum of cotton rats after i.m. and i.d. vaccinations. Vaccination
with protamine-formulated mRNA induces only weak responses.
Vaccination schedule see Table 10. Further details are provided in
Example 2.
[0795] FIG. 2 shows that LNP-formulated mRNA encoding RSV F-protein
(F-del_DSCav1) reduces RSV infection of the lung in cotton rats
after i.m. and i.d. vaccination. Vaccination schedule see Table 10.
Further details are provided in Example 2.
[0796] FIG. 3 shows the results of a lung histopathology analysis
from the RSV cotton rat challenge study described in Example 2.
Animals vaccinated with LNP-formulated mRNA do not show increased
lung histopathology/enhanced inflammation after viral challenge in
contrast to the vaccination with formalin-inactivated RSV virus
vaccine. Vaccination schedule see Table 10. Further details are
provided in Example 2.
[0797] FIG. 4 shows that LNP-formulated mRNA encoding RSV F-protein
(F0, F-del, and F-del_DSCav1) induces high (virus neutralization
titers) VNTs in the serum of cotton rats after two i.m.
vaccinations. VNTs determined performed on day 48. Vaccination
schedule see Table 11. Further details are provided in Example
3.
[0798] FIG. 5 shows that LNP-formulated mRNA encoding RSV F-protein
(F0, F-del, and F-del_DSCav1) induces specific humoral immune
responses in cotton rats against the RSV-F protein. The experiment
was performed as described in Example 3 and antibody total IgG
titers were determined by ELISA. RSV-F (formulated in LNP) and
RSV-F-del (formulated in LNP) vaccines induce higher titers of
RSV-F specific IgGs already after prime vaccination (day 28) than
live virus. FIG. 5a: Antibody titers determined on day 49; FIGS. 5b
and 5c: time-dependent IgG titers in serum with 10 .mu.g mRNA (FIG.
5b) or 100 .mu.g mRNA (FIG. 5c) encoding RSV F-protein (F, F-del,
and F-del_DSCav1). Vaccination schedule see Table 11. Further
details are provided in Example 3.
[0799] FIG. 6 shows that LNP-formulated mRNA encoding RSV F-protein
(F0, F-del, and F-del_DSCav1) reduces RSV infection of the lung in
cotton rats after i.m. vaccination. The experiment was performed as
described in Example 3. All animal groups vaccinated with mRNA
vaccines showed virus titers below the level of detection
demonstrating protection of vaccinated cotton rats in terms of
viral lung titers. In comparison to the mRNA vaccines the vaccine
based on formalin-inactivated virus was not able to prevent virus
titers in the lung. Vaccination schedule see Table 11. Further
details are provided in Example 3.
[0800] FIG. 7 shows that LNP-formulated mRNA encoding RSV F-protein
(F0, F-del, and F-del_DSCav1) reduces RSV titers in the nose of
cotton rats after i.m. vaccination. The experiment was performed as
described in Example 3. All animal groups vaccinated with mRNA
vaccines show strongly reduced viral titers in the nasal tissue in
viral challenge infection experiments. In comparison to the mRNA
vaccines the vaccine based on formalin-inactivated virus was not
able to reduce nasal virus titers. Vaccination schedule see Table
11. Further details are provided in Example 3.
[0801] FIG. 8 shows the results of a lung histopathology analysis
from the RSV cotton rat challenge study described in Example 3.
Animals vaccinated with LNP-formulated mRNA do not show increased
lung histopathology/enhanced inflammation after viral challenge in
contrast to the vaccination with formalin-inactivated RSV virus
vaccine. Vaccination schedule see Table 11. Further details are
provided in Example 3.
[0802] FIG. 9 shows that UTR combinations according to the
invention increase the expression of RSV F-Protein in vitro. HEK
293T cells were transfected with different Lipofectamine-formulated
mRNA constructs, which all contain the same coding sequence
encoding pre-fusion stabilized truncated RSV-F (F-del_DSCav1) but
use different combinations of 5' and 3'-UTRs (see Table 12). RSV-F
expression was analyzed by flow cytometry. Values show % of the
detected RSV-F signal. Values normalized to 100% according to the
expression of the reference mRNA construct (UTR combination
RPL32/ALB7). Water for injection (WFI) serves as a control. N=2.
Further details are provided in Example 4.
[0803] FIG. 10 shows that the used mRNA constructs encoding RSV
F-protein (FIG. 10a: F0, F-del, F0_DSCav1, F-del_DSCav1,
F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2,
F_DSCav1_mut3, F-del_DSCav1_mut3, FIG. 10b: F0, F-del, F0_DSCav1,
F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut4,
F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5) led to a
detectable intracellular RSV F protein expression as well as to a
detectable protein expression at the cell surface. Further details
are provided in Table 16 and Example 8.
[0804] FIG. 11 shows that all tested LNP-formulated mRNA constructs
encoding different RSV F proteins (F0, F0_DSCav1, F_DSCav1_mut3,
F_DSCav1_mut1, F_DSCav1_mut2, F-del, F-del_DSCav1,
F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) induce
humoral immune responses in cotton rats against the RSV-F protein.
Antibody total IgG titers were determined by ELISA. Vaccination
schedule see Table 17. Further details are provided in Example
9.
[0805] FIG. 12 shows that all LNP-formulated RSV-F (F0, F0_DSCav1,
F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del, F-del_DSCav1,
F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) mRNA
vaccines induced the formation of RSV specific functional
antibodies in cotton rats as shown by high virus neutralizing
antibody titers. Vaccination schedule see Table 17. Further details
are provided in Example 9.
[0806] FIG. 13 shows the lung histopathology analysis from the RSV
cotton rat challenge study. None of the mRNA vaccinated groups
displayed enhanced lung pathology as it is the case for the group
that was vaccinated using the formalin-inactivated RSV vaccine
Vaccination schedule see Table 17. Further details are provided in
Example 9.
[0807] FIG. 14 shows the results of the analysis of lung viral
titers (FIG. 14a) and of nose viral titers (FIG. 14b) in cotton
rats challenged with RSV virus. Vaccination schedule see Table 17.
Further details are provided in Example 9.
[0808] FIG. 15 shows that all tested LNP-formulated mRNA constructs
encoding different RSV F proteins (F-del, F-del_DSCav1_mut2,
F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) induce
humoral immune responses in cotton rats against the RSV-F protein.
Antibody total IgG titers were determined by ELISA. Vaccination
schedule see Table 18. Further details are provided in Example
10.
[0809] FIG. 16 shows that all LNP-formulated RSV-F (F-del,
F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5,
F-del_DSCav1_mut4) mRNA vaccines induced the formation of RSV
specific functional antibodies in cotton rats as shown by high
virus neutralizing antibody titers. The LNP-formulated constructs
for pre-fusion stabilization induce higher or comparable responses
than F-del. Vaccination schedule see Table 18. Further details are
provided in Example 10.
[0810] FIG. 17 shows the lung histopathology analysis from the RSV
cotton rat challenge study. None of the mRNA vaccinated groups
displayed enhanced lung pathology as it is the case for the group
that was vaccinated using the formalin-inactivated RSV vaccine
Vaccination schedule see Table 18. Further details are provided in
Example 10.
[0811] FIG. 18 shows the results of the analysis of lung viral
titers (FIG. 18a) and of nose viral titers (FIG. 18b) in cotton
rats challenged with RSV virus. Vaccination schedule see Table 18.
Further details are provided in Example 10.
[0812] FIG. 19 shows that the used mRNA constructs coding for RSV
matrix protein M, phosphoprotein P, nucleoprotein N and matrix
protein M2-1 protein led to a detectable protein expression using a
rabbit reticulocyte lysate system. Further details are provided in
Table 19 and Example 11.
[0813] FIG. 20 shows the results of the immunogenicity study in
mice (ELISA). The humoral immune responses can be seen in FIG. 20a:
anti-RSV F IgG and in FIG. 20b: anti-RSV F IgG2a. All groups
induced humoral immune responses. In general, the IgG2a titers are
10 times higher than the IgG1 titers, indicating a predominant Th1
response. Vaccination schedule see Table 20. Further details are
provided in Example 12.
[0814] FIG. 21 shows that specific antigen IgGs were detected in
sera of immunized mice indicating that the applied mRNA constructs
are suitable to induce specific humoral immune responses.
Vaccination schedule see Table 20. Further details are provided in
Example 12.
[0815] FIG. 22 shows that all LNP-formulated mRNA vaccines (F, M+F,
P+F, N+F, M2-1+F) induced the formation of RSV specific functional
antibodies in mice as shown by high virus neutralizing antibody
titers. Vaccination schedule see Table 20. Further details are
provided in Example 12.
[0816] FIG. 23 shows that all vaccines containing RSV F, RSV M2-1,
or both surprisingly induced a tissue resident memory T cell (TRM)
response in the lung upon intramuscular immunization (FIG. 23a).
The splenocyte analysis revealed that an immunization with RSV F
and especially RSV M2-1 lead to an increase of antigen-specific
CD8+ and CD4+ T cells secreting IFN-.gamma. and TNF indicating the
induction of a systemic T cell response in addition to a
site-specific response (FIG. 23b and FIG. 23c).
EXAMPLES
[0817] In the following, particular examples illustrating various
embodiments and aspects of the invention are presented. However,
the present invention shall not to be limited in scope by the
specific embodiments described herein. The following preparations
and examples are given to enable those skilled in the art to more
clearly understand and to practice the present invention. The
present invention, however, is not limited in scope by the
exemplified embodiments, which are intended as illustrations of
single aspects of the invention only, and methods which are
functionally equivalent are within the scope of the invention.
Indeed, various modifications of the invention in addition to those
described herein will become readily apparent to those skilled in
the art from the foregoing description, accompanying figures and
the examples below. All such modifications fall within the scope of
the appended claims.
Example 1: Preparation of DNA and mRNA Constructs and Compositions
for In Vitro and In Vivo Experiments
[0818] The present Example provides methods of obtaining the
artificial RNA of the invention as well as methods of generating a
composition or a vaccine of the invention.
[0819] 1.1. Preparation of DNA and mRNA constructs:
[0820] For the present examples, DNA sequences encoding different
RSV F proteins (e.g. F0, F-del, F-del_DSCav1, F-del_DSCav1_mut5,
etc.) were prepared and used for subsequent RNA in vitro
transcription reactions. Said DNA sequences were prepared by
modifying the wild type encoding DNA sequences by introducing a G/C
optimized coding sequence (e.g., "cds opt1") for stabilization.
Sequences were introduced into a pUC19 derived vector to comprise
stabilizing 3'-UTR sequences derived from a 3'-UTR of a gene
selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS,
NDUFA1 and RPS9 and 5'-UTR sequences derived from a 5'-UTR of a
gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4,
NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, additionally comprising, a
stretch of adenosines (e.g. 64A or A100), and a histone-stem-loop
(hSL) structure, and optionally a stretch of 30 cytosines (e.g.
C30) as listed in Table 9.
[0821] The obtained plasmid DNA constructs were transformed and
propagated in bacteria using common protocols known in the art.
Eventually, the plasmid DNA constructs were extracted, purified,
and used for subsequent RNA in vitro transcription (see section
1.2.).
[0822] Alternatively, DNA plasmids prepared according to paragraph
1 are used as DNA template for PCR-based amplification. Eventually,
the generated PCR products are purified and used for subsequent RNA
in vitro transcription (see section 1.3.).
[0823] 1.2. RNA In Vitro Transcription from Plasmid DNA
Templates:
[0824] DNA plasmids prepared according to paragraph 1.1 were
enzymatically linearized using EcoRI or SapI and used for DNA
dependent RNA in vitro transcription using T7 RNA polymerase in the
presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog
(e.g., m7GpppG, m7G(5')ppp(5')(2'OMeA)pG, or
m7G(5')ppp(5')(2'OMeG)pG)) under suitable buffer conditions. The
obtained mRNA constructs were purified using RP-HPLC
(PureMessenger.RTM., CureVac AG, Tubingen, Germany; WO2008/077592)
and used for in vitro and in vivo experiments. RNA for clinical
development (see Example 6) is produced under current good
manufacturing practice e.g. according to WO2016/180430,
implementing various quality control steps on DNA and RNA level.
The generated RNA sequences/constructs are provided in Table 9 with
the encoded F protein and the respective UTR elements indicated
therein. In addition to the information provided in Table 9,
further information relating to specific mRNA construct SEQ-ID NOs
may be derived from the information provided under <223>
identifier provided in the ST.25 sequence listing.
[0825] Alternatively, EcoRI or SapI linearized DNA is used for DNA
dependent RNA in vitro transcription using an RNA polymerase in the
presence of a modified nucleotide mixture (ATP, GTP, CTP,
N(1)-methylpseudouridine (m14) or pseudouridine (.PSI.)) and cap
analog (m7GpppG, m7G(5')ppp(5')(2'OMeA)pG, or
m7G(5')ppp(5')(2'OMeG)pG) under suitable buffer conditions. The
obtained m1.PSI. or .PSI.-modified mRNAs are purified using RP-HPLC
(PureMessenger.RTM., CureVac, Tobingen, Germany; WO2008/077592) and
used for further experiments.
[0826] Some mRNA constructs are in vitro transcribed in the absence
of a cap analog. The cap-structure (cap1) is added enzymatically
using Capping enzymes as commonly known in the art. In short, in
vitro transcribed mRNA is capped using an m7G capping kit with
2'-O-methyltransferase to obtain cap1-capped mRNA. Cap1-capped mRNA
is purified using RP-HPLC (PureMessenger.RTM., CureVac, Tubingen,
Germany; WO2008/077592) and used for further experiments.
[0827] 1.3. RNA In Vitro Transcription from PCR Amplified DNA
Templates:
[0828] Purified PCR amplified DNA templates prepared according to
paragraph 1.1 are transcribed in vitro using DNA dependent T7 RNA
polymerase in the presence of a nucleotide mixture
(ATP/GTP/CTP/UTP) and cap analog (m7GpppG) under suitable buffer
conditions. Alternatively, PCR amplified DNA is transcribed in
vitro using DNA dependent T7 RNA polymerase in the presence of a
modified nucleotide mixture (ATP, GTP, CTP,
N(1)-methylpseudouridine (m14)) and cap analog (m7GpppG) under
suitable buffer conditions. Some mRNA constructs are in vitro
transcribed in the absence of a cap analog and the cap-structure
(cap1) is added enzymatically using capping enzymes as commonly
known in the art e.g. using an m7G capping kit with
2'-O-methyltransferase. The obtained mRNAs are purified e.g. using
RP-HPLC (PureMessenger.RTM., CureVac AG, Tubingen, Germany;
WO2008/077592) and used for in vitro and in vivo experiments.
TABLE-US-00014 TABLE 9 mRNA constructs used in the present examples
RNA 5'-UTR/3'-UTR; 3'-terminal SEQ ID NO: SEQ ID NO: ID Antigen
Name UTR Design Elements (3'-end) RNA Protein R6939/ F0 --/muag;
i-3 A64-N5-C30-histoneSL-N5 475 68 R7003 R3737/ F0 RPL32/ALB7; i-2
A64-N5-C30-histoneSL-N5 466 68 R3938 R6940 F-del --/muag; i-3
A64-N5-C30-histoneSL-N5 890 483 R3738/ F-del RPL32/ALB7; i-2
A64-N5-C30-histoneSL-N5 881 483 R3939 R6808 F0_DSCav1 --/muag; i-3
A64-N5-C30-histoneSL-N5 1259 898 R5453 F-del_DSCav1 --/muag; i-3
A64-N5-C30-histoneSL-N5 1628 1267 R6122 F-del_DSCav1 RPL32/ALB7;
i-2 A64-N5-C30-histoneSL-N5 1620 1267 R4745/ F-del_DSCav1
HSD17B4/ALB7; i-4 A64-N5-C30-histoneSL-N5 8278 1267 R5717 R6771
F_DSCav1_mut1 --/muag; i-3 A64-N5-C30-histoneSL-N5 1997 1636 R6774
F-del_DSCav1_mut1 --/muag; i-3 A64-N5-C30-histoneSL-N5 2366 2005
R6772 F_DSCav1_mut2 --/muag; i-3 A64-N5-C30-histoneSL-N5 2735 2374
R6773 F-del_DSCav1_mut2 --/muag; i-3 A64-N5-C30-histoneSL-N5 3104
2743 R6770 F_DSCav1_mut3 --/muag; i-3 A64-N5-C30-histoneSL-N5 3473
3112 R6775 F-del_DSCav1_mut3 --/muag; i-3 A64-N5-C30-histoneSL-N5
3842 3481 F-del_DSCav1 HSD17B4/PSMB3; a-1 A64-N5-C30-histoneSL-N5
1276 1267 F-del_DSCav1 Ndufa4/PSMB3; a-2 A64-N5-C30-histoneSL-N5
1284 1267 F-del_DSCav1 Slc7a3/PSMB3; a-3 A64-N5-C30-histoneSL-N5
1292 1267 F-del_DSCav1 Nosip/PSMB3; a-4 A64-N5-C30-histoneSL-N5
1300 1267 F-del_DSCav1 Mp68/PSMB3; a-5 A64-N5-C30-histoneSL-N5 1308
1267 F-del_DSCav1 Ubqln2/RPS9; b-1 A64-N5-C30-histoneSL-N5 1316
1267 F-del_DSCav1 ASAH1/RPS9; b-2 A64-N5-C30-histoneSL-N5 1324 1267
F-del_DSCav1 HSD17B4/RPS9; b-3 A64-N5-C30-histoneSL-N5 1332 1267
F-del_DSCav1 HSD17B4/CASP1; b-4 A64-N5-C30-histoneSL-N5 1340 1267
F-del_DSCav1 Nosip/COX6B1; b-5 A64-N5-C30-histoneSL-N5 1348 1267
F-del_DSCav1 Ndufa4/RPS9; c-1 A64-N5-C30-histoneSL-N5 1356 1267
F-del_DSCav1 Nosip/Ndufa1; c-2 A64-N5-C30-histoneSL-N5 1364 1267
F-del_DSCav1 Ndufa4/COX6B1; c-3 A64-N5-C30-histoneSL-N5 1372 1267
F-del_DSCav1 Ndufa4/Ndufa1; c-4 A64-N5-C30-histoneSL-N5 1380 1267
F-del_DSCav1 ATP5A1/PSMB3; c-5 A64-N5-C30-histoneSL-N5 1388 1267
F-del_DSCav1 Rpl31/PSMB3; d-1 A64-N5-C30-histoneSL-N5 1396 1267
F-del_DSCav1 ATP5A1/CASP1; d-2 A64-N5-C30-histoneSL-N5 1404 1267
F-del_DSCav1 Slc7a3/Gnas; d-3 A64-N5-C30-histoneSL-N5 1412 1267
F-del_DSCav1 HSD17B4/Ndufa1; d-4 A64-N5-C30-histoneSL-N5 1420 1267
F-del_DSCav1 Slc7a3/Ndufa1; d-5 A64-N5-C30-histoneSL-N5 1428 1267
F-del_DSCav1 TUBB4B/RPS9; e-1 A64-N5-C30-histoneSL-N5 1436 1267
F-del_DSCav1 Rpl31/RPS9; e-2 A64-N5-C30-histoneSL-N5 1444 1267
F-del_DSCav1 Mp68/RPS9; e-3 A64-N5-C30-histoneSL-N5 1452 1267
F-del_DSCav1 Nosip/RPS9; e-4 A64-N5-C30-histoneSL-N5 1460 1267
F-del_DSCav1 ATP5A1/RPS9; e-5 A64-N5-C30-histoneSL-N5 1468 1267
F-del_DSCav1 ATP5A1/COX6B1; e-6 A64-N5-C30-histoneSL-N5 1476 1267
F-del_DSCav1 ATP5A1/Gnas; f-1 A64-N5-C30-histoneSL-N5 1484 1267
F-del_DSCav1 ATP5A1/Ndufa1; f-2 A64-N5-C30-histoneSL-N5 1492 1267
F-del_DSCav1 HSD17B4/COX6B1; f-3 A64-N5-C30-histoneSL-N5 1500 1267
F-del_DSCav1 HSD17B4/Gnas; f-4 A64-N5-C30-histoneSL-N5 1508 1267
F-del_DSCav1 Mp68/COX6B1; f-5 A64-N5-C30-histoneSL-N5 1516 1267
F-del_DSCav1 Mp68/Ndufa1; g-1 A64-N5-C30-histoneSL-N5 1524 1267
F-del_DSCav1 Ndufa4/CASP1; g-2 A64-N5-C30-histoneSL-N5 1532 1267
F-del_DSCav1 Ndufa4/Gnas; g-3 A64-N5-C30-histoneSL-N5 1540 1267
F-del_DSCav1 Nosip/CASP1; g-4 A64-N5-C30-histoneSL-N5 1548 1267
F-del_DSCav1 Rpl31/CASP1; g-5 A64-N5-C30-histoneSL-N5 1556 1267
F-del_DSCav1 Rpl31/COX6B1; h-1 A64-N5-C30-histoneSL-N5 1564 1267
F-del_DSCav1 Rpl31/Gnas; h-2 A64-N5-C30-histoneSL-N5 1572 1267
F-del_DSCav1 Rpl31/Ndufa1; h-3 A64-N5-C30-histoneSL-N5 1580 1267
F-del_DSCav1 Slc7a3/CASP1; h-4 A64-N5-C30-histoneSL-N5 1588 1267
F-del_DSCav1 Slc7a3/COX6B1; h-5 A64-N5-C30-histoneSL-N5 1596 1267
F-del_DSCav1 Slc7a3/RPS9; i-1 A64-N5-C30-histoneSL-N5 1604 1267
F-del_DSCav1 RPL32(32L4)/ALB7; i-2 A64-N5-C30-histoneSL-N5 1612
1267 R7454 F_DSCav1_mut0 --/muag; i-3 A64-N5-C30-histoneSL-N5 4211
3850 R7455 F-del_DSCav1_mut0 --/muag; i-3 A64-N5-C30-histoneSL-N5
4580 4219 R7458 F_DSCav1_mut4 --/muag; i-3 A64-N5-C30-histoneSL-N5
4949 4588 R7459 F-del_DSCav1_mut4 --/muag; i-3
A64-N5-C30-histoneSL-N5 5318 4957 R7456 F_DSCav1_mut5 --/muag; i-3
A64-N5-C30-histoneSL-N5 5687 5326 R7457 F-del_DSCav1_mut5 --/muag;
i-3 A64-N5-C30-histoneSL-N5 6056 5695 F_DSCav1_mut6 --/muag; i-3
A64-N5-C30-histoneSL-N5 6425 6064 F-del_DSCav1_mut6 --/muag; i-3
A64-N5-C30-histoneSL-N5 6794 6433 F_DSCav1_mut7 --/muag; i-3
A64-N5-C30-histoneSL-N5 7163 6802 F-del_DSCav1_mut7 --/muag; i-3
A64-N5-C30-histoneSL-N5 7532 7171 F_DSCav1_mut8 --/muag; i-3
A64-N5-C30-histoneSL-N5 7901 7540 F-del_DSCav1_mut8 --/muag; i-3
A64-N5-C30-histoneSL-N5 8270 7909 R7595 M --/muag; i-3
A64-N5-C30-histoneSL-N5 10046 9684 R7597 N --/muag; i-3
A64-N5-C30-histoneSL-N5 10496 10134 R7598 M2-1 --/muag; i-3
A64-N5-C30-histoneSL-N5 11545 11183 R7596 P --/muag; i-3
A64-N5-C30-histoneSL-N5 10999 10637
[0829] 1.4. Preparation of an LNP Formulated mRNA Composition:
[0830] Lipid nanoparticles (LNP), cationic lipids, and polymer
conjugated lipids (PEG-lipid) were prepared and tested essentially
according to the general procedures described in WO2015/199952,
WO2017/004143 and WO2017/075531, the full disclosures of which are
incorporated herein by reference. LNP formulated mRNA was prepared
using an ionizable amino lipid (cationic lipid), phospholipid,
cholesterol and a PEGylated lipid. Briefly, cationic lipid compound
of formula III-3, DSPC, cholesterol, and PEG-lipid of formula Va
were solubilized in ethanol at a molar ratio (%) of approximately
50:10:38.5:1.5 or 47.4:10:40.9:1.7. LNPs comprising cationic lipid
compound of formula III-3 and PEG-lipid compound of formula IVa
were prepared at a ratio of mRNA to total Lipid of 0.03-0.04 w/w.
The mRNA was diluted to 0.05 mg/mL to 0.2 mg/mL in 10 mM to 50 mM
citrate buffer, pH4. Syringe pumps were used to mix the ethanolic
lipid solution with the mRNA aqueous solution at a ratio of about
1:5 to 1:3 (vol/vol) with total flow rates above 15 ml/min. The
ethanol was then removed and the external buffer replaced with a
PBS buffer comprising Sucrose by dialysis. Finally, the lipid
nanoparticles were filtered through a 0.2 um pore sterile filter
and the LNP-formulated mRNA composition was adjusted to about 1
mg/ml total mRNA. Lipid nanoparticle particle diameter size was
60-90 nm as determined by quasi-elastic light scattering using a
Malvern Zetasizer Nano (Malvern, UK). For other cationic lipid
compounds mentioned in the present specification, the formulation
process is essentially similar. The obtained LNP-formulated mRNA
composition (1 mg/ml total mRNA) was diluted to the desired target
concentration using Saline before in vivo application.
[0831] 1.5. Preparation of a Protamine Complexed mRNA
Composition:
[0832] mRNA constructs were complexed with protamine prior to use
in in vivo immunization experiments. The mRNA formulation consisted
of a mixture of 50% free mRNA and 50% mRNA complexed with protamine
at a weight ratio of 2:1. First, mRNA was complexed with protamine
by addition of protamine-Ringer's lactate solution to mRNA. After
incubation for 10 minutes, when the complexes were stably
generated, free mRNA was added, and the final concentration was
adjusted with Ringer's lactate solution.
Example 2: Vaccination of Cotton Rats with LNP-Formulated mRNA
Encoding RSV-F and RSV Cotton Rat Challenge Study
[0833] The present Example shows that LNP-formulated mRNA encoding
RSV-F induces strong and functional immune responses in cotton
rats.
[0834] For the development of RSV vaccines the cotton rat is an
accepted animal model, especially for the challenge infection.
Cotton rats that have received formalin-inactivated RSV virus
vaccine preparations respond to RSV infection with enhanced lung
pathology. This allows the evaluation of the safety of a
vaccination in terms of enhanced disease phenomenon.
[0835] To optimize the RSV-specific immune response, mRNA vaccines
encoding the pre-fusion stabilized RSV F protein F-del_DSCav were
prepared according to Example 1, formulated either with LNPs (see
Example 1.4.) or with protamine (see Example 1.5.) and were applied
on days 0 and 28 intramuscularly (i.m.) or intradermally (i.d) with
different doses of RNA as shown in Table 10. Control animals
received a single vaccination at day 0 with 105 pfu live RSV/A2
virus intranasally or two vaccinations at days 0 and 28 with
formalin-inactivated RSV virus (FI-RSV) intramuscularly. Additional
control animals received buffer only. For the present example, a
UTR combination HSD17B4/ALB7 was used, herein referred to as
"i-4".
TABLE-US-00015 TABLE 10 Animal groups and vaccination schedule of
Example 2 Group Cotton rats Treatment mRNA dose Route Volume 1 5
mRNA/LNP R4745 10 .mu.g i.m. 1 .times. 100 uL 2 5 mRNA/LNP R4745 2
.mu.g i.m. 1 .times. 100 uL 3 5 mRNA/LNP R4745 10 .mu.g i.d. 2
.times. 50 uL 4 5 mRNA/LNP R4745 2 .mu.g i.d. 2 .times. 50 uL 5 5
mRNA/protamine R5717 80 .mu.g i.m. 1 .times. 100 uL 6 5
mRNA/protamine R5717 80 .mu.g i.d. 2 .times. 50 uL 7 5 Live RSV/A2
virus 105 pfu i.n. 1 .times. 100 uL 8 5 FI-RSV 1:100 i.m. 1 .times.
100 uL 9 5 Buffer -- i.m 1 .times. 100 uL 10 5 Untreated/uninfected
-- --
[0836] Determination of Virus Neutralization Titers:
[0837] Serum was collected at day 49 and RSV virus neutralization
titers (VNTs) were measured using a plaque reduction neutralization
test (PRNT). Diluted serum samples were incubated with RSV/A2 for 1
hour at room temperature and inoculated in duplicates onto
confluent HEp-2 monolayers in 24 well plates. After one hour
incubation at 37.degree. C. in a 5% CO2 incubator, the wells were
overlayed with 0.75% Methylcellulose medium. After 4 days of
incubation, the overlays were removed and the cells were fixed and
stained. The corresponding reciprocal neutralizing antibody titers
were determined at the 60% reduction end-point of the virus
control.
[0838] Cotton Rat Challenge Study:
[0839] The vaccinated animals were challenged intranasally at day
63 with 105 pfu live RSV/A2 virus in 100 uL. One control group
remained untreated and uninfected (group 10). All animals were
sacrificed at day 68 and nasal tissue and lung were harvested.
[0840] RSV Titers in the Lungs of Challenged Cotton Rats:
[0841] Lungs of animals were collected at day 68 (i.e. 5 days after
intranasal challenge with 105 pfu live RSV/A2 virus) and RSV/A2
titers were quantified in one lobe by plaque assay. Lung
homogenates were clarified by centrifugation and diluted in EMEM.
Confluent HEp-2 monolayers were infected in duplicates with diluted
homogenates in 24 well plates. After one hour incubation at
37.degree. C. in a 5% CO incubator, the wells were overlayed with
0.75% methylcellulose medium. After 4 days of incubation, the
overlays were removed and the cells were fixed and stained with
0.1% crystal violet for one hour and then rinsed and air dried.
Plaques were counted and virus titers were expressed as plaque
forming units per gram of tissue. Viral titers were calculated as
geometric mean.+-.standard error for all animals in a group at a
given time.
[0842] Lung Histopathology of Challenged Cotton Rats:
[0843] Lungs of animals were collected at day 68 (i.e. 5 days after
intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe
was analyzed by histopathology. Lungs were dissected and inflated
with 10% neutral buffered formalin to their normal volume, and then
immersed in the same fixative solution. Following fixation, the
lungs were embedded in paraffin, sectioned and stained with
hematoxylin and eosin (H&E). Four parameters of pulmonary
inflammation were evaluated: peribronchiolitis (inflammatory cell
infiltration around the bronchioles), perivasculitis (inflammatory
cell infiltration around the small blood vessels), interstitial
pneumonia (inflammatory cell infiltration and thickening of
alveolar walls), and alveolitis (cells within the alveolar spaces).
Slides were scored blindly on a 0-4 severity scale. The scores were
subsequently converted to a 0-100% histopathology scale.
[0844] Results:
[0845] As can be seen from FIG. 1, the LNP formulated RSV-F
(F-del_DSCav1) mRNA vaccines induce the formation of RSV specific
functional antibodies in cotton rats as shown by high virus
neutralizing antibody titers (groups 1-4). Vaccination with
protamine-formulated mRNA induces only weak responses.
[0846] As can be seen from FIG. 2, the LNP-formulated RSV-F
(F-del_DSCav1) mRNA vaccines reduce lung viral titers in cotton
rats challenged with RSV virus and therefore limit RSV infection of
the lung (groups 1-4). All the animal groups vaccinated with mRNA
formulated in LNPs vaccines showed virus titers below the level of
detection of the performed virus titration demonstrating protection
of vaccinated cotton rats in terms of viral lung titers. By
contrast, the protamine-formulated mRNA vaccines (administrated
i.m.) and the Formalin-inactivated virus vaccine reduced only
minimally the lung virus titer compared to the buffer control
group.
[0847] As can be seen from FIG. 3, the lung histopathology analysis
from the RSV cotton rat challenge study reveals different pathology
scores for the various animal groups. From the histopathology it
can be concluded that none of the mRNA vaccinated groups displayed
enhanced lung pathology as it is the case for the group that was
vaccinated using the formalin-inactivated RSV vaccine. The average
pathology scores for peribronchiolitis, perivasculitis,
insterstitial pneumonia and alveolitis are much lower for all
groups vaccinated with mRNA (groups 1-6) compared to the group with
formalin-inactivated RSV (group 8).
Example 3: Vaccination of Cotton Rats with LNP-Formulated mRNA
Encoding RSV-F and RSV Cotton Rat Challenge Study
[0848] The present Example shows that LNP-formulated mRNA encoding
different RSV-F antigens induce strong and functional immune
responses in cotton rats for mRNA constructs having UTR combination
RPL32/ALB7 (i-2).
[0849] This experiment compared mRNA-LNPs encoding three different
RSV-F variants: full-length RSV F0, truncated RSV F-del, and
pre-fusion stabilized truncated F-del_DSCav1. The mRNA-LNP vaccines
were prepared according to Example 1. Cotton rats received two
intramuscular vaccinations with mRNA-LNP vaccines on days 0 and 28.
Each dose comprised 10 .mu.g or 100 .mu.g mRNA-LNPs. Control
animals received a single vaccination at day 0 with 105 pfu live
RSV/A2 virus intranasally or two vaccinations at days 0 and 28 with
formalin-inactivated RSV virus (FI RSV) intramuscularly. Additional
control animals received buffer only (see Table 11).
TABLE-US-00016 TABLE 11 Animal groups and vaccination schedule of
Example 3 RNA Group Test item ID Dose Route 1 mRNA encoding F0
R3737 10 .mu.g i.m. LNP-formulated 2 mRNA encoding F0 R3737 100
.mu.g i.m. LNP-formulated 3 mRNA encoding F-del R3738 10 .mu.g i.m.
LNP-formulated 4 mRNA encoding F-del R3738 100 .mu.g i.m.
LNP-formulated 5 mRNA encoding F-delDSCav1 R6122 10 .mu.g i.m.
LNP-formulated 6 mRNA encoding F-delDSCav1 R6122 100 .mu.g i.m.
LNP-formulated 7 Live RSV/A2 virus 105 pfu i.n. 8 FI RSV 1:100 i.m.
9 Buffer -- i.m. 10 untreated/uninfected -- --
[0850] Determination of Anti-RSV F Protein Antibodies by ELISA:
[0851] Blood samples were collected on days 28, 49, and 63 for the
determination of anti-RSV F antibody titers. ELISA plates are
coated with recombinant human RSV fusion glycoprotein
(Extracellular domain of human RSV Fusion glycoprotein (529) fused
with a polyhistidine tag at the C-terminus, expressed in
Baculovirus-Insect Cells, provided as lyophilized powder). Coated
plates are incubated using given serum dilutions. Binding of
specific antibodies to the F protein is detected using biotinylated
isotype specific anti-mouse antibodies in combination with
streptavidin-HRP (horse radish peroxidase) with ABTS substrate.
[0852] Determination of Virus Neutralization Titers:
[0853] Serum was collected at day 49 and RSV virus neutralization
titers (VNTs) were measured using a plaque reduction neutralization
test (PRNT). Diluted serum samples were incubated with RSV/A2 for 1
hour at room temperature and inoculated in duplicates onto
confluent HEp-2 monolayers in 24 well plates. After one hour
incubation at 37.degree. C. in a 5% CO2 incubator, the wells were
overlayed with 0.75% Methylcellulose medium. After 4 days of
incubation, the overlays were removed and the cells were fixed and
stained. The corresponding reciprocal neutralizing antibody titers
were determined at the 60% reduction end-point of the virus
control.
[0854] Cotton Rat Challenge Study:
[0855] The vaccinated animals were challenged intranasally at day
63 with 10.sup.5 pfu live RSV/A2 virus in 100 uL. One control group
remained untreated and uninfected. All animals were sacrificed at
day 68 and nasal tissue and lung were harvested.
[0856] RSV Titers in the Lungs of Challenged Cotton Rats:
[0857] Lungs of animals were collected at day 68 (i.e. 5 days after
intranasal challenge with 10.sup.5 pfu live RSV/A2 virus) and
RSV/A2 titers were quantified in one lobe by plaque assay as
described above.
[0858] RSV Titers in Nasal Tissue of Challenged Cotton Rats
[0859] Nasal tissue of animals was collected at day 68 (i.e. 5 days
after intranasal challenge with 105 pfu live RSV/A2 virus). The
nose homogenates were clarified as the lung homogenates by
centrifugation and diluted in EMEM. Confluent HEp-2 monolayers were
infected in duplicates with diluted homogenates in 24 well plates.
After one hour incubation at 37.degree. C. in a 5% CO incubator,
the wells were overlayed with 0.75% methylcellulose medium. After 4
days of incubation, the overlays were removed and the cells were
fixed and stained with 0.1% crystal violet for one hour and then
rinsed and air dried. Plaques were counted and virus titers were
expressed as plaque forming units per gram of tissue. Viral titers
were calculated as geometric mean.+-.standard error for all animals
in a group at a given time.
[0860] Lung Histopathology of Challenged Cotton Rats:
[0861] Lungs of animals were collected at day 68 (i.e. 5 days after
intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe
was analyzed by histopathology as described before.
[0862] Results:
[0863] As can be seen from FIG. 4, all LNP-formulated RSV-F (F0,
F-del, F-del_DSCav1) mRNA vaccines induced the formation of RSV
specific functional antibodies in cotton rats as shown by high
virus neutralizing antibody titers.
[0864] FIGS. 5a, 5b and 5c show that LNP-formulated mRNA encoding
the RSV F-protein (F0, F-del, and F-del_DSCav1) induces humoral
immune responses in cotton rats against the RSV-F protein. Antibody
total IgG titers were determined by ELISA. FIG. 5a: Antibody titers
determined on day 49; FIGS. 5b and 5c: time-dependent igG titers in
serum with 10 .mu.g mRNA (FIG. 5b) or 100 .mu.g mRNA (FIG. 5c)
encoding the RSV F-protein (F0, F-del, and F-del_DSCav1). With both
doses, 10 .mu.g and 100 .mu.g, F0 and F-del vaccines induce higher
titers of RSV-F specific IgGs already after prime vaccination (day
28) compared with the vaccination with the live virus. After boost
vaccination all tested LNP-formulated RSV-F (F0, F-del,
F-del_DSCav1) mRNA vaccines induces humoral immune responses
measured with ELISA which are more prominent then the answers of
the control vaccinations.
[0865] As can be seen from FIG. 6, LNP-formulated RSV-F (F0, F-del,
F-del_DSCav1) mRNA vaccines reduced lung viral titers in cotton
rats challenged with RSV virus and therefore limit RSV infection of
the lung. All the animal groups vaccinated with mRNA formulated in
LNPs vaccines showed virus titers below the level of detection of
the performed virus titration. The results demonstrate protection
of vaccinated cotton rats in terms of viral lung titers. By
contrast, the formalin-inactivated virus vaccine (FI RSV) reduced
only minimally the lung virus titer compared to the buffer control
group.
[0866] As can be seen from FIG. 7, the LNP-formulated RSV-F (F0,
F-del, F-del_DSCav1) mRNA vaccines strongly reduced viral titers in
nasal tissue of cotton rats challenged with RSV virus and therefore
reduce RSV infection of the nose. In comparison to the mRNA
vaccines the vaccine based on formalin-inactivated virus (F RSV)
were not able to reduce the nasal virus titers.
[0867] As can be seen from FIG. 8, the lung histopathology analysis
from the RSV cotton rat challenge study reveals different pathology
scores for the various animal groups. From the histopathology it
can be concluded that none of the mRNA vaccinated groups displayed
enhanced lung pathology as it is the case for the group that was
vaccinated using the formalin-inactivated RSV vaccine. The average
pathology scores for peribronchiolitis, perivasculitis,
insterstitial pneumonia and alveolitis are much lower for all
groups vaccinated with mRNA compared to the group with
formalin-inactivated RSV.
[0868] To further improve the efficiency of the mRNA-based vaccine,
several alternative RSV-F mRNA constructs were designed harboring
different UTR combinations to potentially increase translation
efficiency of the mRNA. Those mRNA constructs were tested as can be
seen in the following Example.
Example 4: In Vitro Expression Screen of RSV-F mRNA Constructs
[0869] The present Example shows that the UTR combinations
according to the invention strongly improve the expression
performance of said mRNA constructs compared to a reference mRNA
construct (harboring RPL32/ALB7 UTRs) used e.g. in Example 3 (UTR
combination RPL32/ALB7 (i-2)).
[0870] To further improve the expression performance of RSV-F mRNA,
a screening experiment was conducted to identify advantageous UTR
combinations according to the invention. To determine the protein
expression performance of RSV-F mRNA constructs comprising
different UTR combinations, HEK 293T cells were transfected with
different Lipofectamine-formulated mRNA constructs, which all
contain the same coding sequence encoding pre-fusion stabilized
truncated F-del_DSCav1 but use different 5'- and 3'-UTRs (see Table
12). RSV-F expression was analyzed 24 h after transfection by flow
cytometry.
TABLE-US-00017 TABLE 12 Overview of mRNA constructs with different
UTR combinations used in Example 4 SEQ ID NO: SEQ ID NO: Antigen
UTR Design RNA Protein F-del_DSCav1 HSD17B4/PSMB3; a-1 1276 1267
F-del_DSCav1 Ndufa4/PSMB3; a-2 1284 1267 F-del_DSCav1 Slc7a3/PSMB3;
a-3 1292 1267 F-del_DSCav1 Nosip/PSMB3; a-4 1300 1267 F-del_DSCav1
Mp68/PSMB3; a-5 1308 1267 F-del_DSCav1 Ubqln2/RPS9; b-1 1316 1267
F-del_DSCav1 ASAH1/RPS9; b-2 1324 1267 F-del_DSCav1 HSD17B4/RPS9;
b-3 1332 1267 F-del_DSCav1 HSD17B4/CASP1; b-4 1340 1267
F-del_DSCav1 Nosip/COX6B1; b-5 1348 1267 F-del_DSCav1 Ndufa4/RPS9;
c-1 1356 1267 F-del_DSCav1 Nosip/Ndufa1; c-2 1364 1267 F-del_DSCav1
Ndufa4/COX6B1; c-3 1372 1267 F-del_DSCav1 Ndufa4/Ndufa1; c-4 1380
1267 F-del_DSCav1 ATP5A1/PSMB3; c-5 1388 1267 F-del_DSCav1
Rpl31/PSMB3; d-1 1396 1267 F-del_DSCav1 ATP5A1/CASP1; d-2 1404 1267
F-del_DSCav1 Slc7a3/Gnas; d-3 1412 1267 F-del_DSCav1
HSD17B4/Ndufa1; d-4 1420 1267 F-del_DSCav1 Slc7a3/Ndufa1; d-5 1428
1267 F-del_DSCav1 TUBB4B/RPS9; e-1 1436 1267 F-del_DSCav1
Rpl31/RPS9; e-2 1444 1267 F-del_DSCav1 Mp68/RPS9; e-3 1452 1267
F-del_DSCav1 Nosip/RPS9; e-4 1460 1267 F-del_DSCav1 ATP5A1/RPS9;
e-5 1468 1267 F-del_DSCav1 ATP5A1/COX6B1; e-6 1476 1267
F-del_DSCav1 ATP5A1/Gnas; f-1 1484 1267 F-del_DSCav1 ATP5A1/Ndufa1;
f-2 1492 1267 F-del_DSCav1 HSD17B4/COX6B1; f-3 1500 1267
F-del_DSCav1 HSD17B4/Gnas; f-4 1508 1267 F-del_DSCav1 Mp68/COX6B1;
f-5 1516 1267 F-del_DSCav1 Mp68/Ndufa1; g-1 1524 1267 F-del_DSCav1
Ndufa4/CASP1; g-2 1532 1267 F-del_DSCav1 Ndufa4/Gnas; g-3 1540 1267
F-del_DSCav1 Nosip/CASP1; g-4 1548 1267 F-del_DSCav1 Rpl31/CASP1;
g-5 1556 1267 F-del_DSCav1 Rpl31/COX6B1; h-1 1564 1267 F-del_DSCav1
Rpl31/Gnas; h-2 1572 1267 F-del_DSCav1 Rpl31/Ndufa1; h-3 1580 1267
F-del_DSCav1 Slc7a3/CASP1; h-4 1588 1267 F-del_DSCav1
Slc7a3/COX6B1; h-5 1596 1267 F-del_DSCav1 Slc7a3/RPS9; i-1 1604
1267 F-del_DSCav1 RPL32(32L4)/ALB7; i-2 1612 1267
[0871] Detailed Description of the Transfection and Flow Cytometry
Analysis:
[0872] 293T cells were seeded at a density of 200000 cells/well
(200000 cells/2 ml) in a 6-well plate. Each RNA was complexed with
Lipofectamine2000 at a ratio of 1/1.5 (w/v) for 20 minutes in
Opti-MEM. Lipocomplexed mRNAs were then added to cells for
transfection with 2 .mu.g of RNA per well in a total volume of 500
uL. 4 h post start of transfection the transfection solution was
exchanged for 2000 uL/well of complete medium. Cells were further
maintained at 37.degree. C., 5% CO2 before performing FACS
analysis.
[0873] 24 hours after transfection, expression of antigen of
interest was quantified by FACS analysis using standard procedures.
Briefly, cells were detached (40 mM Tris HCl pH 7.5 150 mM NaCl, 1
mM EDTA in H20; 5 min at RT), washed with PBS, and stained on the
surface with a mouse antibody against the RSV-F (Millipore, Cat:
MAB8262) and a fluorescently labeled goat anti-mouse IgG antibody
(Sigma, Cat: F5262). Cells were resuspended in 100 uL PFEA buffer
(PBS+2% FCS+2 mM EDTA+0.01% NaN3) and analyzed using a BD FACS
Canto II. Live/Dead staining was performed with Aqua fluorescent
reactive dye (Invitrogen).
[0874] The results were compared to the expression from a reference
construct of Example 3 containing the RPL32/ALB7 UTR-combination
(SEQ ID NO: 1612) which was set to a level of 100%. The results of
the analysis are shown in FIG. 9.
[0875] Results
[0876] As can be seen from FIG. 9, the expression performances of
the mRNA constructs comprising UTR combinations according to the
invention were strongly increased compared to the construct
comprising the reference UTR combination (RPL32/ALB7; i-2).
Example 5: Vaccination of Cotton Rats with LNP-Formulated mRNA
Encoding RSV-F and Cotton Rat Challenge Study
[0877] Based on the results of the UTR screen of Example 4, mRNA
constructs having UTR combinations for optimizing mRNA expression
are used in vaccination experiments.
[0878] In this experiment mRNA vaccines encoding RSV-F (F, F-del,
or F-del_DSCav1) with different UTR combinations according to the
invention are compared. The mRNA-LNP vaccines are prepared
according to Example 1. Cotton rats receive two intramuscular
vaccinations with mRNA-LNP vaccines on days 0 and 28. Each dose
comprises 2 .mu.g or 10 .mu.g mRNA-LNPs. Control animals receive a
single vaccination at day 0 with 105 pfu live RSV/A2 virus
intranasally or formalin-inactivated RSV virus intramuscularly.
Additional control animals receive buffer only (see Table 13).
TABLE-US-00018 TABLE 13 Animal groups and vaccination schedule of
Example 5 SEQ ID NO: Group Test Item 5'-UTR/3'-UTR; RNA Dose Route
1 F0, F-del or F-del_DSCav1 Nosip/RPS9; e-4 286, 701 or 1460 10
.mu.g i.m. 2 F0, F-del or F-del_DSCav1 Nosip/RPS9; e-4 286, 701 or
1460 2 .mu.g i.m. 3 F0, F-del or F-del_DSCav1 Ndufa4/CASP1; g-2
367, 782 or 1532 10 .mu.g i.m. 4 F0, F-del or F-del_DSCav1
Ndufa4/CASP1; g-2 367, 782 or 1532 2 .mu.g i.m. 5 F0, F-del or
F-del_DSCav1 Ndufa4/RPS9; c-1 169, 584 or 1356 10 .mu.g i.m. 6 F0,
F-del or F-del_DSCav1 Ndufa4/RPS9; c-1 169, 584 or 1356 2 .mu.g
i.m. 7 F0, F-del or F-del_DSCav1 Ndufa4/PSMB3; a-2 88, 503 or 1284
10 .mu.g i.m. 8 F0, F-del or F-del_DSCav1 Ndufa4/PSMB3; a-2 88, 503
or 1284 2 .mu.g i.m. 9 F0, F-del or F-del_DSCav1 HSD17B4/PSMB3; a-1
79, 494 or 1276 10 .mu.g i.m. 10 F0, F-del or F-del_DSCav1
HSD17B4/PSMB3; a-1 79, 494 or 1276 2 .mu.g i.m. 11 F0, F-del or
F-del_DSCav1 --/muag; i-3 475, 890 or 1628 10 .mu.g i.m. (R6939,
R6940 or R5453) 12 F0, F-del or F-del_DSCav1 --/muag; i-3 475, 890
or 1628 2 .mu.g i.m. (R6939, R6940 or R5453) 13 Live RSV/A2 virus
5.0Log IM i.n. 14 UV-Inact. RSV/A2 5.0Log IM i.m. 15 FI-RSV 1:100
i.m. 16 Buffer i.m. 17 untreated/uninfected
[0879] Determination of Anti-RSV Immune Responses Using ELISA or
PRNT
[0880] The induction of anti-RSV immune responses are determined as
described before.
[0881] Cotton Rat Challenge Study
[0882] The vaccinated animals are challenged intranasally at day 63
with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group
remains untreated and uninfected. All animals are sacrificed at day
68 and nasal tissue and lung tissue are harvested.
[0883] RSV Titers in the Lungs of Challenged Cotton Rats
[0884] Lungs of animals are collected at day 68 (i.e. 5 days after
intranasal challenge with 10exp5 pfu live RSV/A2 virus) and RSV/A2
titers are quantified in one lobe by plaque assay as described
above. In addition, RSV viral genome copy numbers (by measuring
copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were
determined by quantitative reverse transcription polymerase chain
reaction (qRT-PCR).
[0885] RSV Titers in Nasal Tissue of Challenged Cotton Rats
[0886] Nasal tissue of animals are collected at day 68 (i.e. 5 days
after intranasal challenge with 105 pfu live RSV/A2 virus). The
viral titers are quantified as described above. In addition, RSV
viral genome copy numbers (by measuring copy numbers of the RSV
NS-1 gene) and cytokine mRNA levels were determined by quantitative
reverse transcription polymerase chain reaction (qRT-PCR).
[0887] Lung Histopathology of Challenged Cotton Rats
[0888] Lungs of animals are collected at day 68 (i.e. 5 days after
intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe
is analyzed by histopathology as described before.
Example 6: Vaccination of Cotton Rats with LNP-Formulated mRNA
Encoding RSV-F and Cotton Rat Challenge Study
[0889] In this experiment mRNA vaccines encoding RSV-F
(F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2, F-del_DSCav1_mut3 (and/or additionally with
F_DSCav1_mut0, _mut4, _mut5, _mut6, _mut7, _mut8 and/or
F-del_DSCav1_mut0, _mut4, _mut5, _mut6, _mut7, _mut8) are compared.
The mRNA-LNP vaccines are prepared according to Example 1. Cotton
rats receive two intramuscular vaccinations with mRNA-LNP vaccines
on days 0 and 28. Each dose comprises 2 .mu.g or 10 .mu.g
mRNA-LNPs. Control animals receive a single vaccination at day 0
with 105 pfu live RSV/A2 virus intranasally or formalin-inactivated
RSV virus intramuscularly. Additional control animals receive
buffer only (see Table 14).
TABLE-US-00019 TABLE 14 Animal groups of Example 6 RNA SEQ ID NO:
SEQ ID NO: Group Test Item ID RNA Protein Route 1 F_DSCav1_mut1
R6771 1997 1636 i.m. 2 F-del_DSCav1_mut1 R6774 2366 2005 i.m. 3
F_DSCav1_mut2 R6772 2735 2374 i.m. 4 F-del_DSCav1_mut2 R6773 3104
2743 i.m. 5 F_DSCav1_mut3 R6770 3473 3112 i.m. 6 F-del_DSCav1_mut3
R6775 3842 3481 i.m. 7 Live RSV/A2 virus i.n. 8 UV-Inact. RSV/A2
i.m. 9 FI-RSV i.m. 10 Buffer i.m. 11 Untreated/uninfected
[0890] Determination of Anti-RSV Immune Responses Using ELISA or
PRNT
[0891] The induction of anti-RSV immune responses are determined as
described before.
[0892] Cotton Rat Challenge Study
[0893] The vaccinated animals are challenged intranasally at day 63
with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group
remains untreated and uninfected. All animals are sacrificed at day
68 and nasal tissue and lung tissue are harvested.
[0894] RSV Titers in the Lungs of Challenged Cotton Rats
[0895] Lungs of animals are collected at day 68 (i.e. 5 days after
intranasal challenge with 10exp5 pfu live RSV/A2 virus) and RSV/A2
titers are quantified in one lobe by plaque assay as described
above. In addition, RSV viral genome copy numbers (by measuring
copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were
determined by quantitative reverse transcription polymerase chain
reaction (qRT-PCR).
[0896] RSV Titers in Nasal Tissue of Challenged Cotton Rats
[0897] Nasal tissue of animals are collected at day 68 (i.e. 5 days
after intranasal challenge with 105 pfu live RSV/A2 virus). The
viral titers are quantified as described above. In addition, RSV
viral genome copy numbers (by measuring copy numbers of the RSV
NS-1 gene) and cytokine mRNA levels were determined by quantitative
reverse transcription polymerase chain reaction (qRT-PCR).
[0898] Lung Histopathology of Challenged Cotton Rats Lungs of
animals are collected at day 68 (i.e. 5 days after intranasal
challenge with 10.sup.5 pfu live RSV/A2 virus) and one lobe is
analyzed by histopathology as described before.
Example 7: Vaccination of Cotton Rats with SNP-Formulated mRNA
Encoding RSV-F and a Further RSV Antigen and Cotton Rat Challenge
Study
[0899] To broaden and optimize the RSV-specific immune response,
especially to increase T-cell dependent immune responses, mRNA
vaccines encoding different RSV proteins (RSV F (F0, F, F-del,
F_DSCav1 F-del_DSCav1, or F_DSCav1 mut0-mut8 or F-del_DSCav1
mut0-mut8) are prepared according to Example 1. In order to assess
the effect of single or combined vaccines, these vaccines are
administered either alone or in combination as shown in Table 9.
Cotton rats receive two intramuscular (i.m.) vaccinations with
mRNA-LNP vaccines on days 0 and 28 with 2 .mu.g or 10 .mu.g mRNA
for each antigen. Additional groups are immunized intramuscularly
(i.m.) with formalin-inactivated RSV and/or live RSV/A2 to compare
their immunogenicity to mRNA vaccines. Additional control animals
receive buffer only (see Table 15). The RSV-specific immune
responses are determined by Elisa or Intracellular cytokine
staining (ICS).
TABLE-US-00020 TABLE 15 Animal groups of Example 7 SEQ ID NO: Group
Test Item RNA Route 1 F 475, 890, 1259, 1628, 1997, 2366, 2735,
3104, 3473, 3842, 4211, i.m. 4580, 4949, 5318, 5687, 6056, 6425,
6794, 7163, 7532, 7901, or 8270 2 M 10046 i.m. 3 N 10496 i.m. 4
M2-1 11545 i.m. 5 P 10999 i.m. 6 F + M F (SEQ ID NOs: 475, 890,
1259, 1628, 1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211, 4580,
4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + M
(SEQ ID NO: 10046) 7 F + N F (SEQ ID NOs: 475, 890, 1259, 1628,
1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211, 4580, 4949, 5318,
5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + N (SEQ ID NO:
10496) 8 F + M2-1 F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366,
2735, 3104, i.m. 3473, 3842, 4211,4580, 4949, 5318, 5687, 6056,
6425, 6794, 7163, 7532, 7901, or 8270) + M2-1 (SEQ ID NO: 11545) 9
F + P F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104,
i.m. 3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056, 6425, 6794,
7163, 7532, 7901, or 8270) + P (SEQ ID NO: 10999) 10 F + M + P F
(SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, i.m.
3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163,
7532, 7901, or 8270) + P (SEQ ID NO: 10999) + M (SEQ ID NO: 10046).
11 F + M + N + P F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366,
2735, 3104, i.m. 3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056,
6425, 6794, 7163, 7532, 7901, or 8270) + P (SEQ ID NO: 10999) + M
(SEQ ID NO: 10046) + N (SEQ ID NO: 10496) 12 Live RSV/A2 virus i.n.
13 UV-Inact. RSV/A2 i.m. 14 FI-RSV i.m. 15 Buffer i.m. 16
Untreated/uninfected
[0900] For vaccination experiments of Example 7, polyvalent mRNA
compositions/vaccines are produced (e.g. groups 6-10) according to
procedures as disclosed in the PCT application WO2017/109134 using
at least two different sequence optimized DNA templates (each of
which generated as described in Example 1). In short, a DNA
construct mixture (each of which comprising a different coding
sequences and a T7 promotor) is used as a template for simultaneous
RNA in vitro transcription to generate a mixture of mRNA
constructs. Subsequently, the obtained RNA mixture is used for
co-purification using RP-HPLC. Following that, the obtained
purified RNA mixture is formulated in LNPs (as described in section
1.4.) to generate a polyvalent LNP-formulated RNA
composition/vaccine.
[0901] Determination of Anti-RSV Immune Responses Using ELISA
PRNT:
[0902] The induction of anti-RSV immune responses are determined as
described before, with suitable recombinant proteins or peptides
for its detection.
[0903] Intracellular Cytokine Staining (ICS):
[0904] Splenocytes from vaccinated and control mice are isolated
according to a standard protocol. Briefly, isolated spleens are
grinded through a cell strainer and washed in PBS/1% FBS followed
by red blood cell lysis. After an extensive washing step with
PBS/1% FBS splenocytes are seeded into 96-well plates (2.times.106
cells/well). The next day cells are stimulated with a suitable RSV
peptide or an irrelevant control peptide and 2.5 .mu.g/ml of an
anti-CD28 antibody (BD Biosciences) for 6 hours at 37.degree. C. in
the presence of the mixture of GolgiPlug.TM./GolgiStop.TM. (Protein
transport inhibitors containing Brefeldin A and Monensin,
respectively; BD Biosciences). After stimulation cells are washed
and stained for intracellular cytokines using the Cytofix/Cytoperm
reagent (BD Biosciences) according to the manufacturer's
instructions. The following antibodies are used for staining:
CD8-PECy7 (1:200), CD3-FITC (1:200), IL2-PerCP-Cy5.5 (1:100),
TNF.alpha.-PE (1:100), IFN.gamma.-APC (1:100) (eBioscience), CD4-BD
Horizon V450 (1:200) (BD Biosciences) and incubated with Fcy-block
diluted 1:100. Aqua Dye is used to distinguish live/dead cells
(Invitrogen). Cells are collected using a Canto I flow cytometer
(Beckton Dickinson). Flow cytometry data are analysed using FlowJo
software (Tree Star, Inc.).
[0905] Cotton Rat Challenge Study:
[0906] The vaccinated animals are challenged intranasally at day 63
with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group
remains untreated and uninfected. All animals are sacrificed at day
68 and nasal tissue and lung tissue are harvested.
[0907] Cotton Rat Challenge Study:
[0908] The vaccinated animals are challenged intranasally at day 63
with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group
remains untreated and uninfected. All animals are sacrificed at day
68 and nasal tissue and lung tissue are harvested. Serum is
collected at day 49 and RSV virus neutralization titers (VNTs) are
measured using a plaque reduction neutralization test (PRNT) as
described in Example 2.
[0909] RSV Titers in the Lungs of Challenged Cotton Rats:
[0910] Lungs of animals are collected at day 68 (i.e. 5 days after
intranasal challenge with 10exp5 pfu live RSV/A2 virus) and RSV/A2
titers are quantified in one lobe by plaque assay as described
above. In addition, RSV viral genome copy numbers (by measuring
copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were
determined by quantitative reverse transcription polymerase chain
reaction (qRT-PCR).
[0911] RSV Titers in Nasal Tissue of Challenged Cotton Rats:
[0912] Nasal tissue of animals are collected at day 68 (i.e. 5 days
after intranasal challenge with 105 pfu live RSV/A2 virus). The
viral titers are quantified as described above. In addition, RSV
viral genome copy numbers (by measuring copy numbers of the RSV
NS-1 gene) and cytokine mRNA levels were determined by quantitative
reverse transcription polymerase chain reaction (qRT-PCR).
[0913] Lung Histopathology of Challenged Cotton Rats:
[0914] Lungs of animals are collected at day 68 (i.e. 5 days after
intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe
is analyzed by histopathology as described before.
Example 8: Expression of Different RSV F Proteins in HeLa Cells and
Analysis by FACS
[0915] To determine in vitro protein expression of the mRNA
constructs, HeLa cells were transiently transfected with mRNA
encoding RSV F antigens and stained for RSV Fusing a suitable
anti-F protein antibodies (raised in mouse), counterstained with a
FITC-coupled secondary anti-mouse antibody (F5262 from Sigma). HeLa
cells were seeded in a 6-well plate at a density of 400,000
cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine,
1% Pen/Strep), 24 h prior to transfection. HeLa cells were
transfected with 2 .mu.g unformulated mRNA using Lipofectamine 2000
(Invitrogen). The mRNA constructs prepared according to Example 1
and listed in Table 16 were used in the experiment (see also Table
9), including a negative control (water for injection). 24 hours
post transfection, HeLa cells were stained with suitable anti RSV-F
antibodies (raised in mouse; 1:500) and anti-mouse FITC labelled
secondary antibody (1:500) and subsequently analyzed by flow
cytometry (FACS) on a BD FACS Canto II using the FACS Diva
software. Quantitative analysis of the fluorescent FITC signal was
performed using the FlowJo software package (Tree Star, Inc.).
Cells were stained intracellularly or alternatively on the cell
surface. The results are shown in FIGS. 10a and 10b, representing
two different independent experiments.
TABLE-US-00021 TABLE 16 Overview of mRNA constructs used in Example
8 RNA SEQ ID NO: SEQ ID NO: see ID Antigen UTR Design RNA Protein
Figure R6939 F0 --/muag; i-3 475 68 10a, 10b R6808 F0_DSCav1
--/muag; i-3 1259 898 10a, 10b R6770 F_DSCav1_mut3 --/muag; i-3
3473 3112 10a R6771 F_DSCav1_mut1 --/muag; i-3 1997 1636 10a R6772
F_DSCav1_mut2 --/muag; i-3 2735 2374 10a R6940 F-del --/muag; i-3
890 483 10a, 10b R5453 F-del_DSCav1 --/muag; i-3 1628 1267 10a, 10b
R6775 F-del_DSCav1_mut3 --/muag; i-3 3842 3481 10a R6774
F-del_DSCav1_mut1 --/muag; i-3 2366 2005 10a R6773
F-del_DSCav1_mut2 --/muag; i-3 3104 2743 10a R7454 F_DSCav1_mut0
--/muag; i-3 4211 3850 10b R7456 F_DSCav1_mut5 --/muag; i-3 5687
5326 10b R7458 F_DSCav1_mut4 --/muag; i-3 4949 4588 10b R7455
F-del_DSCav1_mut0 --/muag; i-3 4580 4219 10b R7457
F-del_DSCav1_mut5 --/muag; i-3 6056 5695 10b R7459
F-del_DSCav1_mut4 --/muag; i-3 5318 4957 10b
[0916] Results:
[0917] The results show that the used mRNA constructs led to a
detectable intracellular RSV F protein expression as well as to a
detectable protein expression at the cell surface. The RSV F
constructs with deleted C-terminus (F-del) (e.g. R6940, R5453,
R6775, R6774, R6773, R7455, R7457, R7459) show an increased cell
surface protein expression in comparison to F full length
constructs. The construct encoding F-del_DSCav1_mut0 (R7455)
exhibits the highest expression.
[0918] The results exemplify that the inventive mRNA encoding F
proteins (F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0,
F-del_DSCav1_mut0, F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2,
F-del_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4,
F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5) is translated
in cells and are present at the cell surface, which is a
prerequisite for an mRNA-based RSV vaccine.
Example 9: Vaccination of Cotton Rats with LNP-Formulated mRNA
Encoding RSV-F and RSV Cotton Rat Challenge Study
[0919] The present Example shows that LNP-formulated mRNA according
to the invention encoding different pre-fusion stabilized RSV-F
antigens induce strong, functional and protective immune responses
in cotton rats.
[0920] This experiment compared mRNA-LNPs encoding different
pre-fusion stabilized RSV-F variants (mut 1, mut2, mut3) with
either full-length F or with the truncated F (F-del). The mRNA-LNP
vaccines were prepared according to Example 1. Cotton rats received
two intramuscular vaccinations with mRNA-LNP vaccines on days 0 and
28. Each dose comprised 100 .mu.g mRNA-LNPs. Control animals
received a single vaccination at day 0 with 105 pfu live RSV/A2
virus or UV inactivated RSV/A2 virus or two vaccinations at days 0
and 28 with formalin-inactivated RSV virus (F1 RSV)
intramuscularly. Additional control animals received buffer only
(see Table 17).
TABLE-US-00022 TABLE 17 Animal groups and vaccination schedule of
Example 9 RNA SEQ ID NO: SEQ ID NO: Group Test item ID RNA Protein
Dose Route 1 mRNA encoding F0 R6939 475 68 100 .mu.g i.m.
LNP-formulated 2 mRNA encoding R6808 1259 898 100 .mu.g i.m.
F0_DSCav1 LNP-formulated 3 mRNA encoding R6770 3473 3112 100 .mu.g
i.m. F_DSCav1_mut3 LNP-formulated 4 mRNA encoding R6771 1997 1636
100 .mu.g i.m. F_DSCav1_mut1 LNP-formulated 5 mRNA encoding R6772
2735 2374 100 .mu.g i.m. F_DSCav1_mut2 LNP-formulated 6 mRNA
encoding F-del R6940 890 483 100 .mu.g i.m. LNP-formulated 7 mRNA
encoding R5453 1628 1267 100 .mu.g i.m. F-del_DSCav1 LNP-formulated
8 mRNA encoding R6775 3842 3481 100 .mu.g i.m. F-del_DSCav1_mut3
LNP-formulated 9 mRNA encoding R6774 2366 2005 100 .mu.g i.m.
F-del_DSCav1_mut1 LNP-formulated 10 mRNA encoding R6773 3104 2743
100 .mu.g i.m. F-del_DSCav1_mut2 LNP-formulated 11 Live RSV/A2
virus 10.sup.5 i.m. 12 UV-inact. RSV/A2 10.sup.5 i.m. 13 FI RSV
1:100 i.m. 14 Buffer i.m. 15 untreated/uninfected i.m.
[0921] Determination of Anti-RSV F Protein Antibodies by ELISA:
[0922] Blood samples were collected on days 0, 28, 49, and 63 for
the determination of anti-RSV F antibody titers. ELISA plates were
coated with purified F protein extracted from RSV/A2-infected HEp-2
cells or methanol/Acetone fixed RSV/B infected HEp-2 cells. Coated
plates were incubated using given serum dilutions. Binding of
specific antibodies to the F protein was detected using isotype
specific anti-cotton rat antibodies in combination with
streptavidin-HRP (horse radish peroxidase) with TMB substrate.
[0923] Determination of Virus Neutralization Titers:
[0924] Serum was collected on days 0, 28, 49, and 63 and RSV virus
neutralization titers (VNTs) were measured using a plaque reduction
neutralization test (PRNT). Diluted serum samples were incubated
with RSV/A2 or RSV/B (25-50 PFU) for 1 hour at room temperature and
inoculated in duplicates onto confluent HEp-2 monolayers in 24 well
plates. After one hour incubation at 37.degree. C. in a 5% CO2
incubator, the wells were overlayed with 0.75% Methylcellulose
medium. After 4 days of incubation, the overlays were removed and
the cells were fixed and stained. The corresponding reciprocal
neutralizing antibody titers were determined at the 60% reduction
end point of the virus control.
[0925] Cotton Rat Challenge Study:
[0926] The vaccinated animals were challenged intranasally at day
63 with 10.sup.5 pfu live RSV/A2 virus in 100 uL. One control group
remained untreated and uninfected. All animals were sacrificed at
day 68 and nasal tissue and lung were harvested.
[0927] RSV Titers in Nasal Tissue of Challenged Cotton Rats
[0928] Lungs and nasal tissue of animals were collected at day 68
(i.e. 5 days after intranasal challenge with 10.sup.5 pfu live
RSV/A2 virus). The nose homogenates were clarified as the lung
homogenates by centrifugation and diluted in EMEM. Confluent HEp-2
monolayers were infected in duplicates with diluted homogenates in
24 well plates. After one hour incubation at 37.degree. C. in a 5%
CO incubator, the wells were overlayed with 0.75% methylcellulose
medium. After 4 days of incubation, the overlays were removed and
the cells were fixed and stained with 0.1% crystal violet for one
hour and then rinsed and air dried. Plaques were counted and virus
titers were expressed as plaque forming units per gram of
tissue.
[0929] Pulmonary Histopathology
[0930] Lungs were dissected and inflated with 10% neutral buffered
formalin to their normal volume, and then immersed in the same
fixative solution. Following fixation, the lungs were embedded in
paraffin, sectioned and stained with hematoxylin and eosin
(H&E). Four parameters of pulmonary inflammation were
evaluated: peribronchiolitis (inflammatory cell infiltration around
the bronchioles), perivasculitis (inflammatory cell infiltration
around the small blood vessels), interstitial pneumonia
(inflammatory cell infiltration and thickening of alveolar walls),
and alveolitis (cells within the alveolar spaces). Slides are
scored blind on a 0-4 severity scale. The scores are subsequently
converted to a 0-100% histopathology scale.
[0931] Results:
[0932] FIG. 11 shows that all tested LNP-formulated mRNA constructs
encoding different RSV F proteins (F0, F0_DSCav1, F_DSCav1_mut3,
F_DSCav1_mut1, F_DSCav1_mut2, F-del, F-del_DSCav1,
F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) according
the invention induce humoral immune responses in cotton rats
against the RSV-F protein. Antibody total IgG titers were
determined by ELISA.
[0933] The mRNA constructs encoding the truncated F (F-del) (F-del,
F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2) induced higher and faster responses than the
constructs with the full length F sequence on d28, no significant
difference is detectable at later time points. Stabilized
constructs (F0_DSCav1, F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2,
F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2) initially yield lower ELISA titers than wildtype
constructs (F0 and F-del). Levels are not significantly different
at later time points.
[0934] With a dose of 100 .mu.g, the constructs encoding the
stabilized and truncated F protein (F-del_DSCav1_mut3,
F-del_DSCav1_mut1, F-del_DSCav1_mut2) induce higher titers of RSV-F
specific IgGs already after prime vaccination (day 28) compared
with the vaccination with the live virus. After boost vaccination
all tested LNP-formulated RSV-F mRNA vaccines induces humoral
immune responses measured with ELISA which are more prominent then
the answers of the control vaccinations.
[0935] As can be seen from FIG. 12, all LNP-formulated RSV-F (F0,
F0_DSCav1, F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del,
F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2) mRNA vaccines induced the formation of RSV
specific functional antibodies in cotton rats as shown by high
virus neutralizing antibody titers. The LNP-formulated RSV-F
truncated constructs (F-del, F-del_DSCav1, F-del_DSCav1_mut3,
F-del_DSCav1_mut1, F-del_DSCav1_mut2) induced higher and faster
responses than F full length constructs. The constructs for
pre-fusion stabilization according to the invention (F_DSCav1_mut3,
F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2) induce higher responses than F0, F0_DSCav1,
F-del, F-del_DSCav1.
[0936] As can be seen from FIG. 13, the lung histopathology
analysis from the RSV cotton rat challenge study reveals different
pathology scores for the various animal groups. From the
histopathology it can be concluded that none of the mRNA vaccinated
groups displayed enhanced lung pathology as it is the case for the
group that was vaccinated using the formalin-inactivated RSV
vaccine. The average pathology scores for peribronchiolitis are
lower for all groups vaccinated with mRNA compared to the group
with formalin-inactivated RSV. The LNP-formulated RSV-F truncated
constructs (F-del, F-del_DSCav1, F-del_DSCav1_mut3,
F-del_DSCav1_mut1, F-del_DSCav1_mut2) showed in general lower
pathology scores than F full length constructs. The constructs for
pre-fusion stabilization according to the invention (F_DSCav1_mut3,
F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2) yielded better responses (lower pathology
scores) than F0, F0_DSCav1, F-del, F-del_DSCav1.
[0937] As can be seen from FIG. 14a, all in this experiment tested
LNP-formulated RSV-F mRNA vaccines reduced lung viral titers in
cotton rats challenged with RSV virus and therefore limit RSV
infection of the lung. All the animal groups vaccinated with mRNA
formulated in LNPs vaccines showed virus titers below the level of
detection of the performed virus titration. The results demonstrate
protection of vaccinated cotton rats in terms of viral lung titers.
By contrast, the formalin-inactivated virus vaccine (FI RSV) and
the UV-inactivated RSV/A2 virus control reduced only minimally the
lung virus titer compared to the buffer control group.
[0938] As can be seen from FIG. 14b, the LNP-formulated RSV-F mRNA
vaccines strongly reduced viral titers in nasal tissue of cotton
rats challenged with RSV virus and therefore reduce RSV infection
of the nose. In comparison to the mRNA vaccines the vaccine based
on formalin-inactivated virus (FI RSV) and the UV-inactivated
RSV/A2 virus control were not able to reduce the nasal virus
titers. A complete protection of the nose was only achieved by the
pre-fusion stabilization according to the invention (F_DSCav1_mut3,
F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2).
[0939] To conclude, the results show that LNP-formulated RNA
constructs encoding RSV-F truncated proteins (F-del, F-del_DSCav1,
F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) are in
general more immunogenic than RNA constructs encoding for full
length F proteins. Constructs featuring Ds-Cav1 mutations alone
(F0_DSCav1 and F-del_DSCav1) are suboptimal as an mRNA based
vaccine in comparison with the constructs encoding the further
stabilized F proteins according the invention (F_DSCav1_mut3,
F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1,
F-del_DSCav1_mut2).
[0940] To further improve the efficiency of the mRNA-based vaccine,
several alternative RSV-F mRNA constructs were designed harboring
further mutations stabilizing the pre-fusion conformation in
combination with the beneficial truncated F-del protein. Those mRNA
constructs were tested as can be seen in the following Example.
Example 10: Vaccination of Cotton Rats with LNP-Formulated mRNA
Encoding RSV-F and RSV Cotton Rat Challenge Study
[0941] The present Example shows that LNP-formulated mRNA according
to the invention encoding different pre-fusion stabilized RSV-F
antigens induce strong, functional and protective immune responses
in cotton rats.
[0942] The mRNA-LNP vaccines were prepared according to Example 1.
Cotton rats received either one or two intramuscular vaccinations
with mRNA-LNP vaccines on day 0 or on days 0 and 28. Each dose
comprised 100 .mu.g mRNA-LNPs. Control animals received a single
vaccination at day 28 with 10.sup.5 pfu live RSV/A2 virus or
formalin-inactivated RSV virus (FI RSV) intramuscularly. Additional
control animals received buffer only (see Table 18).
TABLE-US-00023 TABLE 18 Animal groups and vaccination schedule of
Example 10 RNA SEQ ID NO: SEQ ID NO: Group Test item ID RNA Protein
Dose Immunization Route 1 (1) mRNA encoding F-del R6940 890 483 100
.mu.g day 0, day 28 i.m. LNP-formulated 2 (2) mRNA encoding R6773
3104 2743 100 .mu.g day 0, day 28 i.m. F-del_DSCav1_mut2
LNP-formulated 3 (3) mRNA encoding R7455 4580 4219 100 .mu.g day 0,
day 28 i.m. F-del_DSCav1_mut0 LNP-formulated 4 (4) mRNA encoding
R7457 6056 5695 100 .mu.g day 0, day 28 i.m. F-del_DSCav1_mut5
LNP-formulated 5 (5) mRNA encoding R7459 5318 4957 100 .mu.g day 0,
day 28 i.m. F-del_DSCav1_mut4 LNP-formulated 6 (1) mRNA encoding
F-del R6940 890 483 100 .mu.g day 28 i.m. LNP-formulated 7 (2) mRNA
encoding R6773 3104 2743 100 .mu.g day 28 i.m. F-del_DSCav1_mut2
LNP-formulated 8 (3) mRNA encoding R7455 4580 4219 100 .mu.g day 28
i.m. F-del_DSCav1_mut0 LNP-formulated 9 (4) mRNA encoding R7457
6056 5695 100 .mu.g day 28 i.m. F-del_DSCav1_mut5 LNP-formulated 10
(5) mRNA encoding R7459 5318 4957 100 .mu.g day 28 i.m.
F-del_DSCav1_mut4 LNP-formulated 11 Live RSV/A2 virus 10.sup.5 day
28 i.m. 12 FI RSV 1:100 day 28 i.m. 13 Buffer day 0, day 28 i.m. 14
untreated/uninfected i.m.
[0943] Determination of Anti-RSV F Protein Antibodies by ELISA and
Virus Neutralization Titers
[0944] Blood samples were collected on days 0, 28, 49, and 63 for
the determination of anti-RSV F antibody titers and RSV virus
neutralization titers (VNTs) measured using the plaque reduction
neutralization test (PRNT) as described before (see Example 9).
[0945] Cotton Rat Challenge Study:
[0946] The vaccinated animals were challenged intranasally at day
63 with 10.sup.5 pfu live RSV/A2 virus in 100 uL. One control group
remained untreated and uninfected. All animals were sacrificed at
day 68 and nasal tissue and lung were harvested. The RSV titers in
nasal tissue of challenged cotton rats and the pulmonary
histopathology are analyzed as described before (see Example
9).
[0947] Results:
[0948] FIG. 15 shows that all tested LNP-formulated mRNA constructs
encoding different RSV F proteins (F-del, F-del_DSCav1_mut2,
F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) according
the invention induce humoral immune responses in cotton rats
against the RSV-F protein. Antibody total IgG titers were
determined by ELISA. RNA constructs encoding stabilized F proteins
(F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5,
F-del_DSCav1_mut4) initially yield lower ELISA titers than the
construct encoding the truncated wildtype F protein, F-del. This
trend is also detectable at later time points by using higher serum
dilutions for the ELISA (data not shown). The IgG response can be
significantly boosted by a second immunization.
[0949] As can be seen from FIG. 16, all LNP-formulated RSV-F
(F-del, F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5,
F-del_DSCav1_mut4) mRNA vaccines induced the formation of RSV
specific functional antibodies in cotton rats as shown by high
virus neutralizing antibody titers. The LNP-formulated constructs
for pre-fusion stabilization according to the invention
(F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5,
F-del_DSCav1_mut4) induce higher or comparable responses than
F-del. The RNA construct R7457 coding for the pre-fusion stabilized
protein F-del_DSCav1_mut5 induces the highest VNTs (comparable to
the best results induced by best pre-fusion stabilized construct in
the previous experiment (Example 9, R6773).
[0950] The lung histopathology analysis shows that none of the mRNA
vaccinated groups displayed enhanced lung pathology as it is the
case for the group that was vaccinated using the
formalin-inactivated RSV vaccine (see FIG. 17a (for
Peribronciolitis and Perivasculitis) and FIG. 17b (for Interstitial
Pneumonia and Alveolitis)). The constructs for pre-fusion
stabilization according to the invention (F-del_DSCav1_mut2,
F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) yielded
better responses (lower pathology scores) than F-del. Animals
received two vaccinations showed a lower pathology score in
comparison to animals only received one vaccination.
[0951] As can be seen from FIG. 18a, all in this experiment tested
LNP-formulated RSV-F mRNA vaccines reduced lung viral titers in
cotton rats challenged with RSV virus and therefore limit RSV
infection of the lung. This is true even for groups receiving only
one vaccination. The results demonstrate protection of vaccinated
cotton rats in terms of viral lung titers. By contrast, the
formalin-inactivated virus vaccine (FI RSV) reduced only minimally
the lung virus titer compared to the buffer control group.
[0952] As can be seen from FIG. 18b, the LNP-formulated RSV-F mRNA
vaccines strongly reduced viral titers in nasal tissue of cotton
rats challenged with RSV virus and therefore reduce RSV infection
of the nose. In comparison to the mRNA vaccines the vaccine based
on formalin-inactivated virus (FI RSV) was not able to reduce the
nasal virus titers. A complete protection of the nose was only
achieved for some groups received two vaccinations (e.g. for R7457
(F-del_DSCav_mut4) and R7459 (F-del_DSCav_mut5)).
[0953] To conclude, the results show that LNP-formulated RNA
constructs encoding pre-fusion stabilized RSV-F truncated proteins
(F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5,
F-del_DSCav1_mut4) raised in general higher functional titers and
achieved a more pronounced protection against infection in
comparison with the RNA construct coding for the wildtype truncated
F protein F_del. The best results were reached by RNA constructs
encoding F-del_DSCav1_mut2 or F-del_DSCav1_mut5 (R6773, R7457).
Example 11: Expression of Different RSV T-Cell Antigens Using a
Rabbit Reticulocyte Lysate System
[0954] To determine in vitro protein expression of the mRNA
constructs according to the second aspect of the invention (mRNA
constructs coding for RSV matrix protein M, phosphoprotein P,
nucleoprotein N and matrix protein M2-1 protein), unformulated RNAs
(prepared according to Example 1) were mixed with components of
Promega Rabbit Reticulocyte Lysate System according to manufacture
protocol. The lysate contains the cellular components necessary for
protein synthesis (tRNA, ribosomes, amino acids, initiation,
elongation and termination factors). As positive control Luciferase
RNA from Lysate System Kit was used. The translation result was
analyzed by SDS-Page and Western Blot analysis (IRDye 800CW
streptavidin antibody (1:2000)). Table 19 summarizes the tested RNA
constructs.
TABLE-US-00024 TABLE 19 Overview of mRNA constructs used in Example
11 RNA SEQ ID NO: SEQ ID NO: Predicted ID Antigen UTR Design RNA
Protein kDa R7595 M --/muag; i-3 10046 9684 R7596 P --/muag; i-3
10999 10637 27.1 R7597 N --/muag; i-3 10496 10134 43.4 R7598 M2-1
--/muag; i-3 11545 11183 22.1 Luc 61
[0955] Results:
[0956] The results (see FIG. 19) show that the used mRNA constructs
led to a detectable protein expression, which is a prerequisite for
an mRNA-based RSV vaccine (size shifts of RSV P and M2-1 on the gel
may be due to phosphorylation as described e.g. in Richard et al.,
2018 or in Hardy and Wertz, 2000 PMIDs: 29489893 and 10846068).
Example 12: Vaccination of Mice with LNP-Formulated mRNA Encoding
Different RSV T-Cell Antigens Alone and in the Combination with RSV
F (F-Del)
[0957] The present Example analyses LNP-formulated mRNA encoding
different RSV T-cell antigens according to the second aspect of the
invention (RSV M, P, N, and M2-1) alone or in combination with LNP
formulated mRNA encoding the truncated RSV F (F-del). The mRNA-LNP
vaccines were prepared according to Example 1. Balb/c mice received
two intramuscular vaccinations with mRNA-LNP vaccines on days 0 and
28. Each dose comprised 5 .mu.g, 2.5 .mu.g or 2.5 .mu.g+2.5 .mu.g
mRNA-LNPs (for groups receiving mRNAs encoding different antigens
the LNPs were separately formulated and mixed before
administration). Control animals received buffer only (see Table
20).). Humoral, as well as cellular immune responses and
neutralizing antibodies were analyzed by ELISA, FACS, PRNT and
ICS.
TABLE-US-00025 TABLE 20 Animal groups and vaccination schedule of
Example 12 RNA SEQ ID NO: SEQ ID NO: Group Test item ID RNA Protein
Dose Route 1 NaCl buffer -- -- -- i.m. 2 M: mRNA encoding RSV M
R7595 10046 9684 5 .mu.g i.m. LNP-formulated 3 P: mRNA encoding RSV
P R7596 10999 10637 5 .mu.g i.m. LNP-formulated 4 N: mRNA encoding
RSV N R7597 10496 10134 5 .mu.g i.m. LNP-formulated 5 M2-1: mRNA
encoding M2-1 R7598 11545 11183 5 .mu.g i.m. LNP-formulated 6 F:
mRNA encoding F-del R6940 890 483 2.5 .mu.g i.m. LNP-formulated 7 F
+ M: mRNA encoding F-del + R7595 + 890 + 483 + 2.5 .mu.g + i.m.
mRNA encoding RSV M R6940 10046 9684 2.5 .mu.g LNP-formulated 8 F +
P: mRNA encoding F-del + R7596 + 890 + 483 + 2.5 .mu.g + i.m. mRNA
encoding RSV P R6940 10999 10637 2.5 .mu.g LNP-formulated 9 F + N:
mRNA encoding F-del + R7597 + 890 + 483 + 2.5 .mu.g + i.m. mRNA
encoding RSV N R6940 10496 10134 2.5 .mu.g LNP-formulated 10 F +
M2-1: mRNA encoding F-del + R7598 + 890 + 483 + 2.5 .mu.g + i.m.
mRNA encoding RSV M2-1 R6940 11545 11183 2.5 .mu.g
LNP-formulated
[0958] Determination of Anti-RSV F Protein Antibodies by ELISA:
[0959] Blood samples were collected on days 0, 14, 28, and 49 for
the determination of anti-RSV F antibody titers. ELISA plates were
coated with recombinant human RSV fusion glycoprotein (hRSV F
Protein (A2; His-tag; Sino Biological; Cat: 11049-V08B)). Coated
plates were incubated using given serum dilutions. Binding of
specific antibodies to the F protein was detected using isotype
specific anti-mouse rat antibodies in combination with
streptavidin-HRP (horse radish peroxidase) and the Amplex.RTM.
UltraRed Reagent (Invitrogen (Ref. No. A36006).
[0960] Detection of Antigen-Specific Humoral Antibody Responses
Measured by FAGS
[0961] Hela cells were transfected with 2 .mu.g RSV mRNA constructs
(R7595, R7596, R7597, R7598) using lipofectamine. The cells were
harvested 20 h post transfection, and seeded at 1.times.10.sup.5
per well into a 96 well plate. The cells were incubated with
corresponding sera of vaccinated mice (serum of day 28 and 49,
diluted 1:50) followed by a FITC-conjugated anti-mouse IgG antibody
staining. Cells were acquired on BD FACS Canto II using DIVA
software and analyzed by FlowJo. The results are shown in FIG. 21a
(day 28) and FIG. 21b (day 49). As read out the percentage of
FITC-positive cells (out of live cells) was used.
[0962] Determination of Virus Neutralization Titers:
[0963] Serum was collected on day 49 and RSV virus neutralization
titers (VNTs) were measured using a plaque reduction neutralization
test (PRNT). Diluted serum samples were incubated with RSV/A2
(25-50 PFU) for 1 hour at room temperature and inoculated in
duplicates onto confluent HEp-2 monolayers in 24 well plates. After
one hour incubation at 37.degree. C. in a 5% CO2 incubator, the
wells were overlayed with 0.75% Methylcellulose medium. After 4
days of incubation, the overlays were removed and the cells were
fixed and stained. The corresponding reciprocal neutralizing
antibody titers were determined at the 60% reduction end-point of
the virus control.
[0964] Determination of Cellular Immune Responses in Mice by ICS
Using FACS
[0965] Splenocytes and lung cells from vaccinated and control mice
were isolated according to standard protocols. Briefly, isolated
spleens were grinded through a cell strainer and washed in PBS (1%
FCS). After centrifugation, the cell pellet was resuspended with
the remaining supernatant, treated with red cell lysis buffer and
washed repeatedly with PBS (1% FCS). Finally, splenocytes were
resuspended in FCS/10% DMSO, transferred into a cryovial and stored
at -80.degree. C. Lungs were perfused using RPMI-1640 medium (10%
FCS; 2 mM glutamine; 1% Pen/Strep), harvested, and then stored in
RPMI-1640 medium (10% FCS; 2 mM glutamine; 1% Pen/Strep) at
4.degree. C. until lung cells were isolated using the Lung
Dissociation Kit mouse from Miltenyi Biotec according to the
manufacturer's instructions. For ICS, 2.times.10.sup.6 cells
(splenocytes or lung cells) were seed in duplicates in round-bottom
plates and stimulated with RSV M2-1-specific (VYNTVISYI, SYIGSINNI,
SYIESNRKN, KSIDTLSEI), RSV F-specific (FYQSTCSAV, TYMLTNSEL,
KYKNAVTEL, KIMTSKTDV), RSF M-specific (KHTATRFAI, AQMPSKFTI,
KYIKPQSQF, TYLRSISVR), RSV P-specific (SPITSNSTI, SYEEINDQT), or
RSV N-specific (SYKKMLKEM, FYHILNNPK, KYTIQRSTG, GYHVKANGV)
peptides. After Golgi block (GolgiPlug 1/1000) and incubation for
intracellular cytokine accumulation, cells were subjected to
live/dead staining using Aqua-dye. Then, cells were surface stained
using the antibodies .alpha.-CD8-APC-H7 (1/100),
.alpha.-CD4-BD-Horizon V450 (1/200), .alpha.-Thy1.2-FITC (1/300),
and .alpha.-CD107a PE-Cy7 (1/100) for splenocytes and
.alpha.-CD8-APC-H7 (1/100), .alpha.-CD4-BD-Horizon V450 (1/200),
.alpha.-Thy1.2-FITC (1/300), .alpha.-CD69-PE (1/100), and
.alpha.-CD103-PE-Cy7 (1/60) for lung cells. After treatment with
Cytofix/Cytoperm, lung cells were intracellularly stained with
.alpha.-IFNg-APC (1/100) and splenocytes additionally with
.alpha.-TNF.alpha.-PE (1/100). Finally, cells were acquired on BD
FACS Canto II using DIVA software and analyzed by FlowJo.
[0966] Results:
[0967] The results of the immunogenicity study in mice, e.g. the
humoral immune responses can be seen in FIG. 20 (FIG. 20a: anti-RSV
F IgG, FIG. 20b: anti-RSV F IgG2a). In general, all groups induced
humoral immune responses without significant differences between
F-del alone and the F-del+ T-cell antigen groups. The addition of
the T-cell antigens M, P, N, and M2-1 to the mRNA vaccine encoding
RSV F-del did not influence or disturb the immune response against
RSV F. These findings indicate that the co-expression of T cell
antigens does not affect antibody responses induced by RSV F. The
immune responses could be boosted by the second immunization (see
responses on day 49). In general, the IgG2a titers are 10 times
higher than the IgG1 titers, indicating a predominant Th1 response.
A Th1-biased immune response is considered to be an important
prerequisite for a potential RSV vaccine, as Th2-biased responses
have been associated with enhanced respiratory disease (ERD) in
animal models.
[0968] As shown in FIG. 21a and FIG. 21b specific antigen IgGs were
detected in sera of immunized mice indicating that the applied mRNA
constructs are suitable to induce specific humoral immune
responses. Furthermore, the results reveal that the induction of
antigen-specific antibody responses for N and M2-1 can already be
detected at day 28, while for M and P immunization a boost
vaccination is necessary. The decreased antigen-specific antibody
responses after RSV F co-immunization are likely due to different
immunization doses (5 .mu.g vs, 2.5 .mu.g).
[0969] As can be seen from FIG. 22, all LNP-formulated mRNA
vaccines (F, M+F, P+F, N+F, M2-1+F) induced the formation of RSV
specific functional antibodies in mice as shown by high virus
neutralizing antibody titers. The serum neutralizing antibody titer
assays against RSV/A2 revealed no significant differences between
the immunization groups.
[0970] As can be seen from FIG. 23 (FIG. 23a lung ICS CD8, FIG.
23b, spleen ICS CD8, FIG. 23c spleen ICS CD4), all vaccines
containing RSV F, RSV M2-1, or both surprisingly induced a tissue
resident memory T cell (T.sub.RM) response in the lung upon
intramuscular immunization, as an increase of CD8.sup.+ T.sub.RM
cells specific for F or especially for M2-1 could be observed. The
induction of a T.sub.RM cell response in the lung might be
favorable for a potential RSV vaccine, since lung T.sub.RM cells
(CD69.sup.+ and CD103.sup.+) are proposed to protect against RSV
infection. The splenocyte analysis revealed that an immunization
with RSV F and especially RSV M2-1 lead to an increase of
antigen-specific CD8.sup.+ and CD4.sup.+ T cells secreting
IFN.gamma. and TNF indicating the induction of a systemic T cell
response in addition to a site-specific response.
[0971] To conclude, the results show that RSV M2-1 induces the most
promising T cell immune responses without affecting antibody
responses induced by RSV F. Thus, a vaccine containing mRNAs
encoding for RSV F in combination with RSV M2-1 might increase the
protective efficacy against RSV infection.
Example 13: Clinical Development of a RSV mRNA Vaccine
Composition
[0972] To demonstrate safety and efficiency of the RSV mRNA vaccine
composition, a clinical trial (phase 1) is initiated. For clinical
development, RNA is used that has been produced under GMP
conditions (e.g. using a procedure as described in
WO2016/180430).
[0973] In the clinical trial, a cohort of healthy human volunteers
is intramuscularly injected with respective LNP formulated vaccine
compositions comprising favorable UTR combinations.
[0974] In order to assess the safety profile of the vaccine
compositions according to the invention, subjects are monitored
after administration (vital signals, vaccination site tolerability
assessments, hematologic analysis).
[0975] The efficacy of the immunization is analyzed by
determination of virus neutralizing titers (VNT) in sera from
vaccinated subjects. Blood samples are collected on day 0 as
baseline and after completed vaccination. Sera are analyzed for
virus neutralizing antibodies.
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available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210170017A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References