U.S. patent application number 17/194312 was filed with the patent office on 2021-07-22 for chemically ligated rnas for crispr/cas9-lgrna complexes as antiviral therapeutic agents.
The applicant listed for this patent is Dr. Minghong Zhong. Invention is credited to Minghong Zhong.
Application Number | 20210222165 17/194312 |
Document ID | / |
Family ID | 1000005481957 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210222165 |
Kind Code |
A1 |
Zhong; Minghong |
July 22, 2021 |
Chemically Ligated RNAs for CRISPR/Cas9-lgRNA Complexes as
Antiviral Therapeutic Agents
Abstract
Provided herein are chemically ligated guide RNA
oligonucleotides (lgRNA) which comprise two functional RNA modules
(crgRNA and tracrgRNA) joined by non-nucleotide chemical linkers
(nNt-linker), their complexes with CRISPR-Cas9, and cells
comprising lgRNAs, preparation methods of Cas9-1gRNA complexes, and
their uses for prevention and treatments of viral infections in
humans. Also disclosed are processes and methods for preparation of
these compounds.
Inventors: |
Zhong; Minghong;
(Burlingame, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dr. Minghong Zhong |
Burlingame |
CA |
US |
|
|
Family ID: |
1000005481957 |
Appl. No.: |
17/194312 |
Filed: |
March 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15950268 |
Apr 11, 2018 |
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17194312 |
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15006131 |
Jan 26, 2016 |
10059940 |
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15950268 |
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62108064 |
Jan 27, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/30 20130101;
C12N 9/96 20130101; C12N 15/111 20130101; C12N 2310/3519 20130101;
C12N 2310/10 20130101; C12N 2310/20 20170501; A61K 38/00 20130101;
C12N 15/1132 20130101; A61K 31/7105 20130101; C12N 15/11 20130101;
A61K 38/1709 20130101 |
International
Class: |
C12N 15/11 20060101
C12N015/11; A61K 38/17 20060101 A61K038/17; A61K 31/7105 20060101
A61K031/7105; C12N 15/113 20060101 C12N015/113; C12N 9/96 20060101
C12N009/96 |
Claims
1. A cell treated with a composition comprising at least one lgRNA,
and said lgRNA comprises: a. a synthetic crRNA, comprising a spacer
and a second oligonucleotide, wherein i. said spacer is an
oligonucleotide of greater than 12 bases that targets a DNA
sequence, and said second oligonucleotide is an RNA segment of 8-25
nucleotides, ii. said spacer and said second oligonucleotide are
joined between the 3'-end of said spacer and the 5'-end of said
second oligonucleotide via an internucleotide phosphate diester, a
thiophosphate diester ##STR00035## a boranophosphate diester
##STR00036## a phosphonoacetate ##STR00037## a thiophosphonoacetate
##STR00038## a phosphoramidate ##STR00039## or a
thiophosphoramidate ##STR00040## and iii. said spacer and said
second oligonucleotide comprise a single or a plurality of
nucleosides or nucleotides modified at sugar moieties selected from
the group consisting of: ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## wherein Q is a natural or modified
nucleobase, b. a synthetic tracrRNA, comprising: i. a single or a
plurality of oligonucleotides, ii. said oligonucleotides of b. i.
are sequentially joined between the 3'-end and the 5'-end via an
internucleotide phosphate diester, a thiophosphate diester
##STR00046## a boranophosphate diester ##STR00047## a
phosphonoacetate ##STR00048## a thiophosphonoacetate ##STR00049## a
phosphoramidate ##STR00050## or a thiophosphoramidate ##STR00051##
or an nNt-Linker set forth as in c., and iii. said oligonucleotides
of b. i. comprise a single or a plurality of nucleosides or
nucleotides modified at sugar moieties, wherein said nucleosides
and nucleotides are selected from the group consisting of II-1 to
11-37, c. one or more nNt-Linkers, comprising: i. an optionally
substituted M core structure of Formula M-1 to M-18: ##STR00052##
##STR00053## ##STR00054## wherein X=O, S, NH, or CH.sub.2,
X.sub.1.dbd.N or CH, X.sub.2.dbd.N or CH, R.sub.M.dbd.H, CH.sub.3,
alkyl, aryl, or heteroaryl, m=0 to 3 and n=0 to 3, ii. two L
linkers, and each said L linker comprises absent or more structures
selected from the group consisting of L-1 to L-24: ##STR00055##
##STR00056## ##STR00057## where m=0 to 16 and n=0 to 16, and
wherein iii. said L linkers and said M core structure are joined as
L-M-L, wherein the two L linkers are the same or different, and
attached to two terminal nucleotides of Formula Nuc-1 to Nuc-19:
##STR00058## ##STR00059## ##STR00060## ##STR00061## wherein the
attached positions are ##STR00062## to L-M-L and ##STR00063## or
##STR00064## to upstream and downstream oligonucleotides,
respectively, and wherein R is H, OH, ##STR00065## CH.sub.2OH,
##STR00066## F, NH.sub.2, OMe, CH.sub.2OMe, OCH.sub.2CH.sub.2OMe,
an alkyl, a cycloalkyl, an aryl, or a heteroaryl, R' is H, OH,
##STR00067## CH.sub.2OH, ##STR00068## F, NH.sub.2, OMe,
CH.sub.2OMe, OCH.sub.2CH.sub.2OMe, an alkyl, a cycloalkyl, an aryl,
or a heteroaryl, and Q is a natural or a non-natural nucleic acid
base, wherein an nNt-Linker of c. joins the 3'-terminal nucleotide
of said synthetic crRNA of a. and the 5'-terminal nucleotide of
said synthetic tracrRNA of b. and said at least one nNt-Linkers of
c. are positioned outside the bound regions of said lgRNA by a
CRSPR-associated-protein.
2. Said cell of claim 1 comprising at least one said lgRNA.
3. Said cell of claim 1, wherein said composition further comprises
a polypeptide selected from the group consisting of a Cas9, a Cas9
with reduced nuclease activity, a Cas9 with nickase activity, a
Cas9 with no nuclease activity, and a fusion protein comprising a
Cas9 domain, wherein the Cas9 domain is capable of binding with the
activating duplex region and wherein the fusion protein further
comprises a domain from a polypeptide other than Cas9.
4. Said cell of claim 3, wherein said polypeptide is replaced with
its encoding nucleic acid.
5. Said cell of claim 1, wherein said composition further
comprising a fusion protein comprising a Cas9 domain, wherein the
Cas9 domain is capable of binding with the activating duplex region
and wherein the fusion protein further comprises a domain from a
polypeptide other than Cas9 and which confers an additional
activity on the site-directed polypeptide selected from the group
consisting of nuclease activity, methyltransferase activity,
demethylase activity, DNA repair activity, DNA damage activity,
deamination activity, dismutase activity, alkylation activity,
depurination activity, oxidation activity, pyrimidine dimer forming
activity, integrase activity, transposase activity, recombinase
activity, polymerase activity, ligase activity, helicase activity,
photolyase activity, glycosylase activity, acetyltransferase
activity, deacetylase activity, kinase activity, phosphatase
activity, ubiquitin ligase activity, deubiquitinating activity,
adenylation activity, deadenylation activity, SUMOylating activity,
deSUMOylating activity, ribosylation activity, deribosylation
activity, myristoylation activity and demyristoylation
activity.
6. Said cell of claim 5, wherein said fusion protein is replaced
with its encoding nucleic acid.
7. Said cell of claim 1, wherein said composition further comprises
one or more DNA repair templates selected from the group consisting
of ssDNA and dsDNA.
8. Said DNA repair template of claim 7 comprising an
oligonucleotide sequence to introduce insertion(s) of one or more
stop codons selected from the group consisting of 5'-(tga)-3',
5'-(taa)-3', 5'-(tag)-3', 5'-(tga-ntga-ntga)-3',
5'-(tga-ntga-ntaa)-3', 5'-(tga-ntga-ntag)-3',
5'-(tga-ntaa-ntga)-3', 5'-(tga-ntaa-ntaa)-3', 5'-(tgantaa-ntag)-3',
5'-(tga-ntga-ntga)-3', 5'-(tga-ntga-ntaa)-3',
5'-(tga-ntga-ntag)-3', 5'-(taa-ntga-ntga)-3',
5'-(taa-ntga-ntaa)-3', 5'-(taa-ntga-ntag)-3',
5'-(taa-ntaa-ntga)-3', 5'-(taa-ntaa-ntaa)-3',
5'-(taa-ntaa-ntag)-3', 5'-(taa-ntga-ntga)-3',
5'-(taa-ntga-ntaa)-3', 5'-(taa-ntga-ntag)-3',
5'-(tag-ntga-ntga)-3', 5'-(tag-ntga-ntaa)-3',
5'-(tag-ntga-ntag)-3', 5'-(tag-ntaa-ntga)-3',
5'-(tag-ntaa-ntaa)-3', 5'-(tag-ntaa-ntag)-3',
5'-(tag-ntga-ntga)-3', 5'-(tag-ntga-ntaa)-3',
5'-(tag-ntga-ntag)-3', wherein n is any nucleotide, and said more
stop codons comprises repetitive said sequence separated by absent
or more nucleotides in between or different said sequences
separated by absent or more nucleotides in between.
9. Said DNA repair template of claim 7 comprising an
oligonucleotide sequence to introduce insertion(s) of one or more
transcription cis-regulatory elements, and said more elements
comprises repetitive sequence separated by absent or more
nucleotides in between or different sequences separated by absent
or more nucleotides in between.
10. Said cell of claim 7, wherein said DNA repair template is
covalently linked to said lgRNA.
11. Said cell of claim 1, wherein said lgRNA is covalently linked
with one or more molecules selected from the group consisting of
fluorescent molecules, PEGs, non-PEG polymers, ligands of cellular
receptors, lipids, oligonucleotides, polysaccharides, glycans,
peptides, aptamers and antibodies to form an lgRNA conjugate, and
the said more molecules can be the same or different.
12. Said cell of claim 1, wherein said composition further
comprises a carrier containing a DNA repair template, and said
carrier is selected from the group consisting of an AAV vector, a
plasmid and a retron.
13. Said cell of claim 1, wherein the cell is selected from the
group consisting of a bacterial cell, an archaeal cell, a plant
cell, an algal cell, a fungal cell, an invertebrate cell, a
vertebrate cell, a mammalian cell, and a human cell.
14. Said cell of claim 1, wherein the cell comprises deactivated
integrated viral DNAs or deactivated pathogenic genes.
15. Said cell of claim 1, wherein the cell comprises deactivated
integrated HBV DNAs.
16. Said cell of claim 1, wherein the cell comprises deactivated
integrated HIV DNAs.
17. Said cell of claim 1, wherein the cell comprises a deactivated
CCR5 gene.
18. Said cell of claim 1, wherein the cell comprises deactivated
integrated HSV DNAs.
19. Said cell of claim 1, wherein the cell comprises an edited host
gene.
20. Said cell of claim 1, wherein the cell comprises one or more
inserted genes.
21. Said cell of claim 1, wherein the cell further comprises other
therapeutic agents.
22. Said cell of claim 2, wherein said at least one lgRNA is
introduced into the cell by lipofection, electroporation,
nucleofection, microinjection, biolistics, in liposomes,
immunoliposomes, with polycations, as nucleic acid conjugates, or
combinations thereof.
23. A cell comprising at least one said ligated tracrRNA of claim
1.
24. Said cell of claim 23, wherein said tracrRNA is covalently
linked to one or more molecules selected from the group consisting
of fluorescent molecules, PEGs, non-PEG polymers, ligands of
cellular receptors, lipids, oligonucleotides, polysaccharides,
glycans, peptides, aptamers and/or antibodies to form a tracrRNA
conjugate, and the said more molecules can be the same or
different.
Description
CROSS REFERENCE TP RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 15/950,268, filed Apr. 11, 2018, which is a
divisional application of U.S. application Ser. No. 15/006,131,
filed Jan. 26, 2016 now U.S. Pat. No. 10,059,940, with title
"Chemically Ligated RNAs for CRISPR/Cas9-lgRNA Complexes as
Antiviral Therapeutic Agents" and naming Minghong Zhong as
inventor(s), and claims the benefits of U.S. Provisional
Application Ser. No. 62/108,064, the entire said invention being
incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to chemically synthesized
guide RNA oligonucleotides (lgRNA) comprising two functional RNA
modules (crgRNA and tracrgRNA) ligated by non-nucleotide chemical
linker(s) (nNt-linker), their complexes with CRISPR-Cas9,
preparation methods of Cas9-lgRNA complexes, and uses as medicinal
agents in treatment of chronic viral infections.
BACKGROUND OF THE INVENTION
[0003] Chronic viral infections such as HIV, HBV, and HSV afflict
enormous suffering, life loss, and financial burdening among the
infected individuals. These infectious diseases are incurable, and
contagious to variable degrees, and are prominent threats for
public health, highlighting the urgent needs for curative
therapies. To date, effective antiviral therapies only suppress
viral replication but do not clear virus in patients, and do not
target the viral genetic materials of latently integrated (proviral
DNA) or non-replicating episomal viral genomes (such as cccDNA) in
human cells. Nevertheless, these viral DNAs have been reported to
directly cause these chronic or latent infections.
[0004] Recent breakthroughs in biotechnology have identified
several molecular gene editing tools such as zinc finger nucleases
(ZFNs), transcription activator-like (TAL) effector nucleases
(TALENs), homing endonucleases (HEs), and most notably the clusters
of regularly interspaced short palindromic repeat
(CRISPR)-associated protein Cas9. These nucleases are highly
specific, DNA-cleaving enzymes, and have been recently demonstrated
as potential therapeutic applications to eradicate HBV, HIV, HSV,
and herpes virus by targeting the enzymatic systems directly to
essential viral genome sequences.
[0005] Both ZFNs and TALENs are composed of protein-based
programmable, sequence-specific DNA-binding modules and nonspecific
DNA cleavage domains, which make multiple-site targeting extremely
challenging. This is even more challenging for HEs, which have the
same protein domains for both DNA binding and cleavage.
[0006] Unlike nucleases such as ZFNs, TALENs, and HEs,
CRISPR-Cas-RNA complexes contain short CRISPR RNAs (crRNAs), which
direct the Cas proteins to the target nucleic acids via
Watson-Crick base pairing to facilitate nucleic acid destruction.
Three types (I-III) of CRISPR-Cas systems have been functionally
identified across a wide range of microbial species. While the type
I and III CRISPR systems utilize ensembles of Cas proteins
complexed with crRNAs to mediate the recognition and subsequent
degradation of target nucleic acids, the type II CRISPR system
recognizes and cleaves the target DNA via the RNA-guided
endonuclease Cas9 along with two noncoding RNAs, the crRNA and the
trans-activating crRNA (tracrRNA). The crRNA::tracrRNA complex
directs Cas9 DNA endonuclease to the protospacer on the target DNA
next to the protospacer adjacent motif (PAM) for site-specific
cleavage (FIG. 1). This system is further simplified by fusing the
crRNA and tracrRNA into a single chimeric guide RNA (sgRNA) with
enhanced efficiency, and can be easily reprogrammed to cleave
virtually any DNA sequence by redesigning the crRNAs or sgRNAs.
[0007] The CRISPR/Cas9 system has been evaluated as potential
therapeutic strategy to cure chronic and/or latent viral infections
such as HIV, HBV, and Epstein-Barr virus (EBV). Hu and et al.
reported CRISPR/Cas9 system could eliminate the integrated HIV-1
genome by targeting the HIV-1 LTR U3 region in single and multiplex
configurations. It inactivated viral gene expression and
replication in latently infected microglial, promonocytic, and T
cells, completely excised a 9,709-bp fragment of integrated
proviral DNA that spanned from its 5' to 3' LTRs, and caused
neither genotoxicity nor off-target editing to the host cells. Yang
and et al. reported the CRISPR/Cas9 system could significantly
reduce the production of HBV core and surface proteins in Huh-7
cells transfected with an HBV-expression vector, and disrupt the
HBV expressing templates both in vitro and in vivo, indicating its
potential in eradicating persistent HBV infection. They observed
that two combinatorial gRNAs targeting different sites could
increase the efficiency in causing indels. The study by Seeger and
Sohn also reported CRISPR/Cas9 efficiently inactivated HBV genes in
NTCP expressing HepG2 cells permissive for HBV infection. Wang and
Quake observed patient-derived cells from a Burkitt's lymphoma with
latent Epstein-Barr virus infection presented dramatic
proliferation arrest and a concomitant decrease in viral load after
exposure to a CRISPR/Cas9 vector targeted to the viral genome and a
mixture of seven guide RNAs at the same molar ratio via
plasmid.
[0008] In spite of initial success in evaluation of CRISPR/Cas9 for
therapeutic applications in treatment of chronic or latent viral
infections, off-target cleavage is a major concern for any nuclease
therapy. Studies indicated that a 10-12 bp "seed" region located at
the 3'-end of the protospacer sequence is critical for its
site-specific cleavage, and that Cas9 can tolerate up to seven
mismatches at the 5'-end of the 20-base protospacer sequence.
[0009] Cas9::sgRNA was reported to be a single turnover enzyme, and
was tightly bound by the cleaved DNA products. In thermodynamic
term, the energetic increase in the unwound DNA helix (two DNA
single strand) in Cas9 is compensated by free energy decrease of
the hybridization between DNA and crRNA and of the binding of Cas9
with the formed DNA:crRNA helix and with the resulting single
strand DNA; to release the cleaved DNA products, especially the
hybridized DNA strand with crRNA, the free energy increase in the
released DNA requires compensation of free energy decrease by
certain processes such as binding of additional protein factor(s),
enabling the recycling of the Cas::sgRNA enzyme, which is believed
to happen under physiological conditions. A more related but also
more complicated in vitro assay comprising both Cas9::sgRNA and
other binding molecules or cellular assay is needed for reliable
SAR based on multiple turnover enzymatic reactions.
[0010] Another major concern is the requirement of more
sophisticated approaches to delivery. To date, the Cas9 protein and
RNAs (either dualRNA (crRNA/tracrRNA) or a single guide RNA (sgRNA,
.about.100 nt)) have been mostly introduced by plasmid transfection
either as whole or separately, which makes chemical modifications
of RNAs extremely challenging, if not impossible, and also is
limited by random integration of all or part of the plasmid DNA
into the host genome and by persistent and elevated expression of
Cas9 in target cells that could lead to off-target effects. A
recent study presented lipid-mediated delivery of unmodified
Cas9::sgRNA complexes using common cationic lipid nucleic acid
transfection reagents such as Lipofectamine resulted in up to 80%
genome modification with substantially higher specificity compared
to DNA transfection, and be effective both in vitro and in vivo.
Another example presented treatment with cell penetrating peptide
(CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide
RNAs led to endogenous gene disruptions in human cell lines with
lower off-target mutation frequencies than plasmid
transfection.
[0011] To minimize or completely eradicate off-target effects, and
to overcome other major obstacles to its pharmaceutical
applications including the lack of stability of RNA, low potency of
Cas9-gRNA at the target sites, large sizes of Cas9 proteins
(.about.150 kDa), chemically modified sgRNAs may provide a very
effective strategy as indicated by the therapeutic application of
antisense oligonucleotides and progress in small interfering RNA
technology, and also supported by a recent report on chemically
modified, 29-nucleotide synthetic CRISPR RNA (scrRNA) in
combination with unmodified transactivating crRNA (tracrRNA),
showing a comparable efficacy as sgRNA, though as dual guide RNAs,
this combination should be less effective than sgRNA incorporated
with same chemical modifications because of entropic and other
factors. However, considering the size of sgRNA (.about.100 nt),
large scale chemically manufacturing such large molecules is
industrially challenging and costly. Methods for preparation of
long RNAs (sgRNA) compatible to extensive chemical modifications
and/or significantly truncated sgRNAs or crRNA/tracrRNAs of lengths
compatible to current industrial RNA chemical synthesis are in
need. Truncating sgRNAs or crRNA/tracrRNAs is limited because of
the many essential binding interactions between Cas9 and sgRNA and
the complicated molecular mechanisms of recognition and cleavage of
target DNAs. A possible solution can be based on recent progress in
chemical ligations of nucleic acids. Brown and et al. showed the
CuAAC reaction (click chemistry) in conjunction with solid-phase
synthesis could produce catalytically active hairpin ribozymes
around 100 nucleotides in length.
[0012] Recent disclosure of the structure of CRISPR/Cas9-sgRNA
complexes indicated that the linkloop (tetraloop) of the sgRNA
protrudes outside of the CRISPR/Cas9-sgRNA complex, with the distal
4 base pairs (bp) completely free of interactions with Cas9 peptide
side chains (FIG. 2). The natural guide RNAs comprise CRISPR RNA
(crRNA) and trans-activating crRNA (tracrRNA), directing Cas9 to a
specific genomic locus harboring the target sequence, in spite of
their lower efficacy than sgRNA. Therefore this tetraloop can be
replaced by small molecule non-nucleotide linkers (nNt-linker), and
sgRNA can be divided into two pieces of .about.30-32 nt (crgRNA)
and .about.60 nt (tracrgRNA) (FIG. 3), respectively, or into three
pieces (.about.30-32 nt (crgRNA), .about.30 nt (tracrgRNA1), and
.about.30 nt (tracrgRNA2)), or multiple pieces based on the void of
interactions between certain other sections of sgRNA and Cas9, and
joined by chemical ligations in vitro as ligated guide RNAs
(lgRNA). These RNAs (crgRNA and tracrgRNA) are more readily
synthesized chemically at industrial scale, and can even be further
shortened by introductions of chemical modifications at various
sites, and therefore more commercially accessible, and lgRNAs can
be optimized by chemical modifications for better efficacy and
specificity and for targeted delivery.
[0013] As presented by previous studies, targeting viral DNA at
multiple sites could enhance the effectiveness, which can be better
practiced by delivering whole CRISPR/Cas9-lgRNA complexes composed
of chemical libraries of different crgRNAs (including various
spacers) and formed in vitro. This chemical ligation strategy can
provide diverse chemically modified lgRNAs targeting multiple sites
and/or variants/mutations of a single site in viral genomes
equivalent to combination therapies such as HAART.
SUMMARY OF THE INVENTION
[0014] This invention pertains to chemically ligated guide RNA
oligonucleotides (lgRNA) comprising two functional RNA modules
(crgRNA and tracrgRNA, or crgRNA and dual-tracrgRNA or
multiple-tracrgRNA) joined by chemical nNt-linkers, their complexes
with CRISPR-Cas9, preparation methods for Cas9-lgRNA complexes and
their uses in the treatments of viral infections in humans,
represented by the following structure (Formula I):
##STR00001##
[0015] wherein
[0016] (a) spacer (NNNNNNNNNNNNN . . . ) is an oligonucleotide
derived from viral genomes corresponding to a viral DNA sequence
(protospacer) immediately before NGG or any of other protospacer
adjacent motifs (PAM);
[0017] (b) spacers (NNNNNNNNNNNNN . . . ) comprise nucleotides
modified at sugar moieties such as 2'-deoxyribonucleotides,
2'-methoxyribonucleotides, 2'-F-ribonucleotides,
2'-F-arabinonucleotides, 2'-O,4'-C-methylene nucleotides (LNA),
unlocked nucleotides (UNA), nucleoside phosphonoacetate (PACE),
thiophosphonoacetate (thioPACE), and phosphoromonothioates:
##STR00002##
wherein Q is a nucleic acid base;
[0018] (c) A . . . B base pair is selected from A . . . U, U . . .
A, C . . . G, and G . . . C;
[0019] (d) "nNt-Linker" is a chemical moiety joining the 3'-end of
crgRNA and 5'-end of tracrgRNA, and is constructed by click
chemistry or chemical ligations; the attaching position at the
3'-end nucleotide of crgRNA can be selected from 3'-, 2'-, and 7-
(for 7-deazapurinenucleoside) or 5-(for pyrimidine nucleosides) or
8-(for purine nucleosides) positions, and the attaching position at
the 5'-end nucleotide of tracrgRNA can be selected from 5'-, 2'-,
and 7-(for 7-deazapurinenucleoside) or 5-(for pyrimidine
nucleosides) or 8-(for purine nucleosides) positions, as
represented by non-limiting examples below:
##STR00003##
[0020] (d) Nucleotides in [ ] ([CU] and [GA]) can be deleted or
replaced with other nucleotides preferably with base pair(s)
unaffected with or without modifications of sugar moieties;
[0021] (e) The Cas9-lgRNA complexes can be prepared in the
following steps (FIG. 4):
[0022] Step 1. Synthesis of crgRNA and tracrgRNA. Structures of
non-limiting examples are given below:
##STR00004##
[0023] Step 2. Chemical ligation of crgRNA and tracrgRNA and
annealing to form RNA duplex, lgRNA:
##STR00005##
or annealing followed by chemical ligation to form lgRNA.
[0024] Step 3. In vitro complex formation between lgRNA and Cas9
protein.
[0025] (f) crgRNA in (e) can be single oligonucleotide or a mixture
of oligonucleotides of various sequences, and thus the preparation
gives a mixture of combinatorial Cas9-lgRNA complexes targeting
multiple sites in viral genomes.
[0026] In some embodiments, tracrgRNA is a single RNA with chemical
modifications or without chemical modifications.
[0027] In some embodiments, tracrgRNA is a ligated dual
oligonucleotide, comprising tracrgRNA1 and tracrgRNA2, or multiple
oligonucleotides.
[0028] Provided herein are chemically ligated oligonucleotides for
CRISPR-Cas9, their complexes with CRISPR-Cas9, preparation methods
and uses of these complexes as medicinal agents in treatment of
viral infections. Embodiments of these structures, preparations,
and applications are described in details herein.
[0029] In certain embodiments, lgRNAs comprise modified nucleotides
in crgRNAs, and non-limiting examples include
2'-deoxyribonucleotides, 2'-methoxyribonucleotides,
2'-F-ribonucleotides, 2'-F-arabinonucleotides,
2'-methoxyethoxyribonucleotides, 2'-0,4'-C-methylene nucleotides
(LNA), unlocked nucleotide analogues (UNA), nucleoside
phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), and
phosphoromonothioates.
[0030] In certain embodiments, lgRNAs comprise modified nucleotides
in tracrgRNAs, and non-limiting examples include
2'-deoxyribonucleotides, 2'-methoxyribonucleotides,
2'-F-ribonucleotides, 2'-F-arabinonucleotides,
2'-methoxyethoxyribonucleotides, 2'-0,4'-C-methylene nucleotides
(LNA), unlocked nucleotide analogues (UNA), nucleoside
phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), and
phosphoromonothioates.
[0031] In certain embodiments, lgRNAs comprise nucleotides modified
in base moieties of tracrgRNAs, and non-limiting examples include
2,6-diaminopurine, 5-fluorouracil, pseudouracil, and
7-fluoro-7-deazapurine.
[0032] In certain embodiments, lgRNAs comprise nucleotides modified
in base moieties of crgRNA, and non-limiting examples include
2,6-diaminopurine, 5-fluorouracil, pseudouracil, and
7-fluoro-7-deazapurine.
[0033] In certain embodiments, lgRNAs comprise modified nucleotides
in both crgRNAs and tracrgRNAs.
[0034] In certain embodiments, lgRNAs comprise nucleotides modified
in both base moieties and sugar moieties.
[0035] In certain embodiments, the nNt-linker comprises a
1,4-disubstituted 1,2,3-triazole formed between an alkyne and an
azide via [3+2] cycloaddition catalyzed by Cu(I) or without Cu(I)
catalysis (such as strain-promoted azide-alkyne cycloaddition
(SPAAC)).
[0036] In certain embodiments, the nNt-linker is a thioether by
chemical ligation between a thiol and a maleimide, or other
functional groups.
[0037] In some embodiments, the 3'-end nucleotide of crgRNA is a
modified U or C, and the nNt-linker is attached at 5-position of
the nucleoside base, or a 7-deazaguanine or 7-deazaadenine, and the
nNt-linker is attached at 7-position of the nucleoside base, or a
purine nucleotide, and the nNt-linker is attached at the 8-position
of the nucleoside base.
[0038] In other embodiments is, and the nNt-linker is attached at
the 2'- or 3'-position of the sugar moiety of the 3'-end nucleotide
of crgRNA.
[0039] In some embodiments, the 5'-end nucleotide of tracrgRNA is a
modified U or C, and the nNt-linker is attached at 5-position of
the nucleoside base, or a 7-deazaguanine or 7-deazaadenine, and the
nNt-linker is attached at 7-position of the nucleoside base, or a
purine nucleotide, and the nNt-linker is attached at the 8-position
of the nucleoside base.
[0040] In other embodiments, the nNt-linker is attached at the 2'-
or 5'-position of the sugar moiety of the 5'-end nucleotide of
tracrgRNA.
[0041] In some embodiments, the 5'-end nucleotide of tracrgRNA is
base-paired with the 3'-end nucleotide of crgRNA as A . . . 0 or G
. . . C.
[0042] In other embodiments, the 5'-end nucleotide of tracrgRNA and
the 3'-end nucleotide of crgRNA are not base-paired.
[0043] In certain embodiments, lgRNAs comprise spacers in crgRNA
selected from sequences of 12.about.20 nt of HIV genomes, of which
each thymine is replaced with uracil, immediately before a PAM
(such as NGG). The spacer RNA oligomers have the same sequences as
the sense strands (5'.fwdarw.3') of the genomes, and namely are
their RNA transcripts with or without further chemical
modifications, or have the same sequences as the antisense strands
(5' .fwdarw.3') of the genomes, and namely are their antisense RNA
transcripts with or without further chemical modifications.
[0044] In certain embodiments, lgRNAs comprise spacers in crgRNA
selected from sequences of 12.about.20 nt of HBV genomes, of which
each thymine is replaced with uracil, immediately before a PAM
(such as NGG). The spacer RNA oligomers have the same sequences as
the sense strands (5'.fwdarw.3') of the genomes, and namely are
their RNA transcripts with or without further chemical
modifications, or have the same sequences as the antisense strands
(5' .fwdarw.3') of the genomes, and namely are their antisense RNA
transcripts with or without further chemical modifications.
[0045] In certain embodiments, lgRNAs comprise spacers in crgRNA
selected from sequences of 12.about.20 nt of HSV genomes, of which
each thymine is replaced with uracil, immediately before a PAM
(such as NGG). The spacer RNA oligomers have the same sequences as
the sense strands (5'.fwdarw.3') of the genomes, and namely their
RNA transcripts with or without further chemical modifications, or
have the same sequences as the antisense strands (5'.fwdarw.3') of
the genomes, and namely are their antisense RNA transcripts with or
without further chemical modifications.
[0046] In certain embodiments, lgRNAs comprise spacers in crgRNA
selected from sequences of 12.about.20 nt of EBV genomes, of which
each thymine is replaced with uracil, immediately before a PAM
(such as NGG). The spacer RNA oligomers have the same sequences as
the sense strands (5'.fwdarw.3') of the genomes, and namely their
RNA transcripts with or without further chemical modifications, or
have the same sequences as the antisense strands (5'.fwdarw.3') of
the genomes, and namely are their antisense RNA transcripts with or
without further chemical modifications.
[0047] In certain embodiments, lgRNAs are mixtures comprising lgRNA
constructs with different spacers corresponding to different loci
of viral genomes and/or variants of a single locus of target
genomes. Useful selection methods to identify sequences having
extremely low to no homology between the foreign viral genome and
host cellular genome including endogenous retroviral DNA include
bioinformatics screening to minimize and/or exclude off-target
human transcriptome and essential untranslated genomic sites, and
to optimize the efficacy of target cleavage.
DETAILED DESCRIPTION OF THE INVENTION
[0048] An aspect of the invention is directed to chemically ligated
guide RNA oligonucleotides (lgRNA) comprising two functional RNA
modules (crgRNA and tracrgRNA, crgRNA and dual-tracrgRNA or
multiple-tracrgRNA) joined by chemical nNt-linkers, their complexes
with CRISPR-Cas9, preparation methods of Cas9-lgRNA complexes and
their uses in the treatments of viral infections in humans.
[0049] In some embodiments, this chemical ligation strategy
provides diverse chemically modified lgRNAs for optimization for
better efficacy in cleaving viral genomic DNAs.
[0050] In other embodiments, this chemical ligation strategy
provides diverse chemically modified lgRNAs for minimizing
off-target cleavages of host genomic DNAs with or without
engineering Cas9 proteins.
[0051] In some embodiments, this chemical ligation strategy
provides diverse chemically modified lgRNAs for decreasing the size
of Cas9-lgRNA complex by engineering Cas9 proteins, or for full
functional substitution of Cas9 with smaller natural/engineered
CRISPR-associated proteins thus more amenable to delivery in human
cells, for efficient administrations and better dosage forms.
[0052] In other embodiments, Cas9-lgRNA complexes are formulated
with transfecting agents such as cationic lipids, cationic polymers
and/or cell penetrating peptides for antiviral therapies, either
alone or in combination with other direct-acting antiviral agents
(DAA).
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1: shows crRNA (Top, SEQ ID NO: 5)::tracrRNA (Bottom,
SEQ ID NO: 6) complex in upper diagram, and schematic illustration
of a Cas9/gRNA complex bound at its target DNA site at the
bottom.
[0054] FIG. 2: shows molecular surface representation of the
crystal structure of a Cas9/gRNA complex, and the tetraloop is free
of any interactions with Cas9.
[0055] FIG. 3: shows a molecular model of lgRNA. The ligation
nNt-linker (ligation1) is a triazole.
[0056] FIG. 4: shows a general procedure to prepare a Cas9-lgRNA
complex.
DEFINITION
[0057] The definitions of terms used herein are consistent to those
known to those of ordinary skill in the art, and in case of any
differences the definitions are used as specified herein
instead.
[0058] The term "nucleoside" as used herein refers to a molecule
composed of a heterocyclic nitrogenous base, containing an
N-glycosidic linkage with a sugar, particularly a pentose. An
extended term of "nucleoside" as used herein also refers to acyclic
nucleosides and carbocyclic nucleosides.
[0059] The term "nucleotide" as used herein refers to a molecule
composed of a nucleoside monophosphate, di-, or triphosphate
containing a phosphate ester at 5'-, 3'-position or both. The
phosphate can also be a phosphonate.
[0060] The term of "oligonucleotide" (ON) is herein used
interchangeably with "polynucleotide", "nucleotide sequence", and
"nucleic acid", and refers to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. An oligonucleotide may comprise one or more
modified nucleotides, which may be imparted before or after
assembly of such as oligonucleotide. The sequence of nucleotides
may be interrupted by non-nucleotide components.
[0061] The term of "crispr/cas9" refers to the type II CRISPR-Cas
system from Streptococcus pyogenes. The type II CRISPR-Cas system
comprises protein Cas9 and two noncoding RNAs (crRNA and tracrRNA).
These two noncoding RNAs were further fused into one single guide
RNA (sgRNA). The Cas9/sgRNA complex binds double-stranded DNA
sequences that contain a sequence match to the first 17-20
nucleotides of the sgRNA and immediately before a protospacer
adjacent motif (PAM). Once bound, two independent nuclease domains
(HNH and RuvC) in Cas9 each cleaves one of the DNA strands 3 bases
upstream of the PAM, leaving a blunt end DNA double stranded break
(DSB).
[0062] The term of "off-target effects" refers to non-targeted
cleavage of the genomic DNA target sequence by Cas9 in spite of
imperfect matches between the gRNA sequence and the genomic DNA
target sequence. Single mismatches of the gRNA can be permissive
for off-target cleavage by Cas9. Off-target effects were reported
for all the following cases: (a) same length but with 1-5 base
mismatches; (b) off-target site in target genomic DNA has one or
more bases missing (`deletions`); (c) off-target site in target
genomic DNA has one or more extra bases (`insertions`).
[0063] The term of "guide RNA" (gRNA) refers to a synthetic fusion
of crRNA and tracrRNA via a tetraloop (GAAA) (defined as sgRNA) or
other chemical linkers such as an nNt-Linker (defined as lgRNA),
and is used interchangeably with "chimeric RNA", "chimeric guide
RNA", "single guide RNA" and "synthetic guide RNA". The gRNA
contains secondary structures of the repeat:anti-repeat duplex,
stem loops 1-3, and the linker between stem loops 1 and 2.
[0064] The term of "dual RNA" refers to hybridized complex of the
short CRISPR RNAs (crRNA) and the trans-activating crRNA
(tracrRNA). The crRNA hybridizes with the tracrRNA to form a
crRNA:tracrRNA duplex, which is loaded onto Cas9 to direct the
cleavage of cognate DNA sequences bearing appropriate
protospacer-adjacent motifs (PAM).
[0065] The term of "lgRNA" refers to guide RNA (gRNA) joined by
chemical ligations to form non-nucleotide linkers (nNt-linkers)
between crgRNA and tracrgRNA, or at other sites.
[0066] The terms of "dual lgRNA", "triple lgRNA" and "multiple
lgRNA" refer to hybridized complexes of the synthetic guide RNA
fused by chemical ligations via non-nucleotide linkers. Dual
tracrgRNA is formed by chemical ligation between tracrgRNA1 and
tracrgRNA2 (RNA segments of .about.30 nt), and crgRNA (.about.30
nt) is fused with a dual tracrgRNA to form a triple lgRNA duplex,
which is loaded onto Cas9 to direct the cleavage of cognate DNA
sequences bearing appropriate protospacer-adjacent motifs (PAM).
Each RNA segment can be readily accessible by chemical
manufacturing and compatible to extensive chemical
modifications.
[0067] The term "guide sequence" refers to the about 20 bp sequence
within the guide RNA that specifies the target site and is herein
used interchangeably with the terms "guide" or "spacer". The term
"tracr mate sequence" may also be used interchangeably with the
term "direct repeat(s)".
[0068] The term of "crgRNA" refers to crRNA equipped with chemical
functions for conjugation/ligation and is used interchangeably with
crRNA in an lgRNA comprising at least one non-Nucleotide linker.
The oligonucleotide may be chemically modified close to its 3'-end,
any one or several nucleotides, or for its full sequence.
[0069] The term of "tracrgRNA" refers to tracrRNA equipped with
chemical functions for conjugation/ligation and is used
interchangeably with tracrRNA in an lgRNA comprising at least one
non-Nucleotide linker. The oligonucleotide may be chemically
modified at any one or several nucleotides, or for its full
sequence.
[0070] The term of "the protospacer adjacent motif (PAM)" refers to
a DNA sequence immediately following the DNA sequence targeted by
Cas9 in the CRISPR bacterial adaptive immune system, including NGG,
NNNNGATT, NNAGAA, NAAAC, and others from different bacterial
species where N is any nucleotide.
[0071] The term of "chemical ligation" refers to joining together
synthetic oligonucleotides via an nNt-linker by chemical methods
such as click ligation (the azide-alkyne reaction to produce a
triazole linkage), thiol-maleimide reaction, and formations of
other chemical groups.
[0072] The term of "complementary" refers to the ability of a
nucleic acid to form hydrogen bond(s) with another nucleic acid
sequence by either traditional Watson-Crick or other
non-traditional types. Cas9 contains two nuclease domains, HNH and
RuvC, which cleave the DNA strands that are complementary and
noncomplementary to the 20 nucleotide (nt) guide sequence in
crRNAs, respectively.
[0073] The term of "Hybridization" refers to a reaction in which
one or more polynucleotides form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
complex may comprise two strands forming a duplex structure, three
or more strands forming a multi stranded complex, a single
self-hybridizing strand, or any combination of these. A sequence
capable of hybridizing with a given sequence is referred to as the
"complement" of the given sequence.
[0074] The synonymous terms "hydroxyl protecting group" and
"alcohol-protecting group" as used herein refer to substituents
attached to the oxygen of an alcohol group commonly employed to
block or protect the alcohol functionality while reacting other
functional groups on the compound. Examples of such
alcohol-protecting groups include but are not limited to the
2-tetrahydropyranyl group, 2-(bisacetoxyethoxy)methyl group, trityl
group, trichloroacetyl group, carbonate-type blocking groups such
as benzyloxycarbonyl, trialkylsilyl groups, examples of such being
trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl,
phenyldimethylsilyl, triiospropylsilyl and thexyldimethylsilyl,
ester groups, examples of such being formyl, (C.sub.1-C.sub.10)
alkanoyl optionally mono-, di- or tri-substituted with
(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkoxy, halo, aryl,
aryloxy or haloaryloxy, the aroyl group including optionally mono-,
di- or tri-substituted on the ring carbons with halo,
(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkoxy wherein aryl is
phenyl, 2-furyl, carbonates, sulfonates, and ethers such as benzyl,
p-methoxybenzyl, methoxymethyl, 2-ethoxyethyl group, etc. The
choice of alcohol-protecting group employed is not critical so long
as the derivatized alcohol group is stable to the conditions of
subsequent reaction(s) on other positions of the compound of the
formula and can be removed at the desired point without disrupting
the remainder of the molecule. Further examples of groups referred
to by the above terms are described by J. W. Barton, "Protective
Groups In Organic Chemistry", J. G. W. McOmie, Ed., Plenum Press,
New York, N.Y., 1973, and G. M. Wuts, T. W. Greene, "Protective
Groups in Organic Synthesis", John Wiley & Sons Inc., Hoboken,
N.J., 2007, which are hereby incorporated by reference. The related
terms "protected hydroxyl" or "protected alcohol" define a hydroxyl
group substituted with a hydroxyl protecting group as discussed
above.
[0075] The term "nitrogen protecting group," as used herein, refers
to groups known in the art that are readily introduced on to and
removed from a nitrogen atom. Examples of nitrogen protecting
groups include but are not limited to acetyl (Ac), trifluoroacetyl,
Boc, Cbz, benzoyl (Bz), N,N-dimethylformamidine (DMF), trityl, and
benzyl (Bn). See also G. M. Wuts, T. W. Greene, "Protective Groups
in Organic Synthesis", John Wiley & Sons Inc., Hoboken, N.J.,
2007, and related publications.
[0076] The term of "Isotopically enriched" refers to a compound
containing at least one atom having an isotopic composition other
than the natural isotopic composition of that atom. The term of
"Isotopic composition" refers to the amount of each isotope present
for a given atom, and "natural isotopic composition" refers to the
naturally occurring isotopic composition or abundance for a given
atom. As used herein, an isotopically enriched compound optionally
contains deuterium, carbon-13, nitrogen-15, and/or oxygen-18 at
amounts other than their natural isotopic compositions.
[0077] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any agent(s) which can be used in the
treatment or prevention of a disorder or one or more symptoms
thereof. In certain embodiments, the term "therapeutic agent"
includes a compound provided herein. In certain embodiments, a
therapeutic agent is an agent known to be useful for, or which has
been or is currently being used for the treatment or prevention of
a disorder or one or more symptoms thereof.
[0078] Nucleotides
[0079] In some embodiments, the crRNA and tracrRNA are truncated at
3'-end and 5'-end, respectively:
##STR00006##
[0080] and the duplex end is replaced with a small molecule
non-nucleotide linker (nNt-linker, ligation1), instead of the
tetraloop (GAAA) in sgRNA, to form a ligated dual lgRNA:
##STR00007##
[0081] wherein "NNNNNNNNNN NNNNNNNNNNNNN" is a guide sequence of
17-20 nt, and N is preferably a ribonucleotide with intact 2'-OH,
and wherein "" is a chemical nNt-linker.
[0082] In other embodiments, tracrgRNA is a ligated dual
oligonucleotide (via ligation2, the inner ligation between
tracrgRNA1 and tracrgRNA2), or a multiple oligonucleotide.
Non-limiting examples include:
##STR00008##
[0083] In some embodiments, the crRNA and tracrRNA are further
truncated, and non-limiting examples of resulting lgRNAs
include:
##STR00009##
[0084] The two RNA modules (crgRNA and tracrgRNA, crgRNA and
dual-tracrgRNA or multiple-tracrgRNA) are synthesized chemically by
phosphoramidite chemistry and ligation chemistry either on solid
support(s) or in solution. Non-limiting examples of compounds
(oligonucleotide azides, oligonucleotide alkynes, oligonucleotide
thiols, and oligonucleotide maleimides) and synthetic methods
include:
[0085] 1. Ligation Via Formation of a 1,2,3-Triazole
Cross-Linker
[0086] a. Direct modification of fully deprotected oligonucleotide
amine (such as ON-1) provides RNA alkynes or azides (ON-2):
##STR00010##
[0087] Non-limiting examples of crosslinking reagents include:
##STR00011##
[0088] b. Azide and alkyne functions are introduced as
phosphoramidites in chemical synthesis of the RNAs (ON-5 and
ON-7):
##STR00012## ##STR00013##
[0089] Non-limiting examples of these phosphoramidites include:
##STR00014## ##STR00015## ##STR00016##
[0090] c. Preparation of lgRNA by chemical ligation via click
chemistry of crgRNA and tracrgRNA and annealing to form RNA duplex,
or by annealing followed by chemical ligation via click
chemistry.
[0091] 2. Ligation Via Formation of a Thioether Cross-Linker
[0092] a. Thiol function is introduced as a phosphoramidite in
chemical synthesis of the RNAs:
##STR00017##
[0093] Non-limiting examples of these phosphoramidites include:
##STR00018##
[0094] b. Direct modification of fully deprotected oligonucleotide
amine (such as ON-1) provides the RNA maleimide (ON-10):
##STR00019##
[0095] Non-limiting examples of maleimide crosslinking reagents
include:
##STR00020##
[0096] c. Preparation of lgRNA by chemical ligation via thio-ether
formation between crgRNA and tracrgRNA and annealing to form RNA
duplex, or by annealing followed by chemical ligation via
thio-ether formation.
[0097] The above chemical ligations for ligation between crgRNA and
tracrgRNA (ligation1) are applicable to formation of ligated dual
tracrgRNA between tracrgRNA1 and tracrgRNA2 (ligation2).
[0098] In some embodiments, the two ligations (ligation1 and
ligation2) are chemically orthogonal. Non-limiting examples
include:
[0099] a. ligation2 by a triazole linker and ligation1 by a
thioether linker;
[0100] b. ligation2 by a thioether linker and ligation1 by a
triazole linker;
[0101] c. ligation2 by some linker and ligation1 by a second
linker, which is chemically orthogonal to that for ligation2;
[0102] In other embodiments, the two ligations can be formed by the
same chemistry such as an azide-alkyne [3+2] cycloaddition.
[0103] An aspect of the invention is directed to CRISPR-Cas9-lgRNA
system:
[0104] wherein lgRNA is a single ligated RNA composed of
dual-modules, a triple or a multiple ligated RNA composed of
dual-modules, and ligation sites are preferably located at
tetraloop (A32-U37), and/or stem 2 (C70-G79) (of reported sgRNA
designed for S. pyogenes Cas9), while any 3', 5'-phosphodiester of
sgRNA can be replaced by single nNt-Linker as a ligation site; and
wherein Cas9 composed of a nuclease lobe (NUC) and a recognition
lobe (REC) can be a CRISPR-associated protein other than the
example of S. pyogenes Cas9 represented here, and can be any
engineered Cas9.
[0105] Some aspects of the invention are directed to
CRISPR-Cas9-lgRNA-transfecting reagent(s) systems.
[0106] Some aspects of the invention are directed to the use of
CRISPR-Cas9-lgRNA system for antiviral therapy targeting against
proviral DNAs or episomal circular DNAs.
[0107] In some embodiments, non-limiting examples of targeted viral
genomic sequences of HBV include:
TABLE-US-00001 (SEQ ID NO 36) ctctgctagatcccagagtg [aGG], (SEQ ID
NO 37) gctatcgctggatgtgtctg [cGG], (SEQ ID NO 38)
tggacttctctcaattttct [a[G[G]G]G]], (SEQ ID NO 39)
gggggatcacccgtgtgtct [tGG], (SEQ ID NO 40) tatgtggatgatgtggtactgg
[gGG], (SEQ ID NO 41) cctcaccatacagcactc [gGG], (SEQ ID NO 42)
gtgttggggtgagttgatgaatc [tGG],
[0108] wherein nucleotides in [ ] are PAMs, or any sequence of
17-20 nt upstream adjacent to a PAM sequence. More examples of such
sequences can be found in literature (Lee, et al.,
WO2016/197132A1). A complete list of such sequences can be found in
viral genomic sequences of HBV using online Basic Local Alignment
Search Tool (BLAST). Useful selection methods to identify sequences
having extremely low to no homology between the foreign viral
genome and host cellular genome including endogenous retroviral DNA
include bioinformatics screening to minimize and/or exclude
off-target human transcriptome and essential untranslated genomic
sites, and to optimize the efficacy of target cleavage.
[0109] In some embodiments, the therapeutic Cas9-lgRNAs comprise
multiple lgRNAs targeting at different sites in viral genome
(multiplex editing).
[0110] In some embodiments, multiple crgRNAs, including but not
limited to sequences of YMDD and its mutations at catalytic domain
of HBV polymerase, corresponding to
TABLE-US-00002 (SEQ ID NO 43) tatgtggatgat gtggtactgg [gGG], (SEQ
ID NO 44) tatatggatgat gtggtattgg [gGG], (SEQ ID NO 45)
tatgtggatgat gtggtattgg [gGG], (SEQ ID NO 46) tatatagatgat
gtggtactgg [gGG],
[0111] are ligated to tracrgRNA to result in a mixture of lgRNAs,
and thus of Cas9-lgRNAs to target drug resistance in therapies
based on direct-acting antiviral agents (DAA).
[0112] In some embodiments, non-limiting examples of targeted viral
genomic sequences of HIV include:
TABLE-US-00003 (SEQ ID NO 47) gattggcaga actacacacc [aGG], (SEQ ID
NO 48) atcagatatc cactgacctt [tGG], (SEQ ID NO 49) gcgtggcctg
ggcgggactg [gGG], (SEQ ID NO 50) cagcagttct tgaagtactc [cGG],
[0113] wherein nucleotides in [ ] are PAMs, or any sequence of
17-20 nt upstream adjacent to a PAM sequence. A complete list of
such sequences can be found in viral genomic sequences of HIV using
online Basic Local Alignment Search Tool (BLAST). Useful selection
methods to identify sequences having extremely low to no homology
between the foreign viral genome and host cellular genome including
endogenous retroviral DNA include bioinformatics screening to
minimize and/or exclude off-target human transcriptome and
essential untranslated genomic sites, and to optimize the efficacy
of target cleavage.
[0114] In some embodiments, non-limiting examples of targeted viral
genomic sequences of herpesviridae virus such as HSV and EBV
include:
TABLE-US-00004 (SEQ ID NO 51) gccctggaccaacccggccc [gGG], (SEQ ID
NO 52) ggccgctgccccgctccggg [tGG], (SEQ ID NO 53)
ggaagacaatgtgccgcca [tGG], (SEQ ID NO 54) tctggaccagaaggctccgg
[cGG], (SEQ ID NO 55) gctgccgcggagggtgatga [cGG], (SEQ ID NO 56)
ggtggcccaccgggtccgct [gGG], (SEQ ID NO 57) gtcctcgagggggccgtcgc
[gGG],
[0115] wherein nucleotides in [ ] are PAMs, or any sequence of
17-20 nt upstream adjacent to a PAM sequence. A complete list of
such sequences can be found in viral genomic sequences of
herpesviridae virus using online Basic Local Alignment Search Tool
(BLAST). Useful selection methods to identify sequences having
extremely low to no homology between the foreign viral genome and
host cellular genome including endogenous retroviral DNA include
bioinformatics screening to minimize and/or exclude off-target
human transcriptome and essential untranslated genomic sites, and
to optimize the efficacy of target cleavage.
[0116] Other aspects of the invention are directed to the use of
CRISPR-Cas9-lgRNA systems for gene edition and therapy.
[0117] Process for Preparations of Nucleotides
[0118] Other embodiments of this invention represent processes for
preparation of these compounds provided herein, which can also be
prepared by any other methods apparent to those skilled in the
art.
[0119] Chemical Ligations
[0120] In some embodiments, the ligation between crgRNA and
tracrgRNA (ligation1) is formation of triazole by Cu(I) catalyzed
[2+3] cycloaddition. An azide or alkyne function is introduced at
3'-end of crgRNA which is an aminoalkyl oligonucleotide as
represented by a non-limiting example of ON-11:
##STR00021##
[0121] An azide or alkyne function is introduced at 5'-end
nucleotide of tracrgRNA as represented by a non-limiting example of
ON-12 by solid phase supported synthesis:
##STR00022##
[0122] TracrgRNA and crgRNA are then ligated by click reaction, and
annealed as represented by a non-limiting example of ON-13:
##STR00023##
or are annealed, and then ligated by click chemistry.
[0123] In other embodiments, the ligation between crgRNA and
tracrgRNA is formation of thioether linker or any compatible
crosslinking chemistry.
[0124] In some embodiments for triple lgRNAs, the ligation in
tracrgRNA (ligation2) and the ligation between crgRNA and tracrgRNA
(ligation1) are orthogonal, and two ligations are realized in one
pot:
[0125] A maleimide is introduced to 3'-end of crgRNA in solid phase
supported synthesis as represented by a non-limiting example of
ON-14:
##STR00024##
[0126] A thiol and an azide are introduced at 5'-end and 3'-end of
tracrgRNA1, respectively as represented by a non-limiting example
of ON-15:
##STR00025##
[0127] An alkyne is introduced at 5'-end of tracrgRNA2 as
represented by a non-limiting example of ON-16:
##STR00026##
[0128] TracrgRNA1, tracrgRNA2, and crgRNA are then ligated, and
annealed as represented by a non-limiting example of ON-17:
##STR00027##
[0129] In some embodiments, the ligation in tracrgRNA (ligation2)
and the ligation between crgRNA and tracrgRNA (ligation1) are
orthogonal, and two ligations are realized in two sequential
steps.
[0130] In other embodiments, the ligation in tracrgRNA (ligation2)
and the ligation between crgRNA and tracrgRNA (ligation1) are
formed by the same ligation chemistry, and two ligations are
realized sequentially as represented by synthesis of ON-20:
##STR00028## ##STR00029##
[0131] Formation OF Cas9-lgRNA, Cellular Transfections, and
ASSAYS
[0132] The formation of Cas9-lgRNA complex, and cellular
transfections are performed as reported.
[0133] a. Transfection with cationic lipids (Liu, D. et al. Nature
Biotechnology 2015, 33, 73-80):
[0134] Purified synthetic lgRNA or mixture of synthetic lgRNAs is
incubated with purified Cas9 protein for 5 min, and then complexed
with the cationic lipid reagent in 25 .mu.L OPTIMEM. The resulting
mixture is applied to the cells for 4 h at 37.degree. C.
[0135] b. Transfection with cell-penetrating peptides (Kim, H. et
al. Genome Res. 2014, 24: 1012-1019):
[0136] Cell-penetrating peptide (CPP) is conjugated to a purified
recombinant Cas9 protein (with appended Cys residue at the C
terminus) by drop wise mixing of 1 mg Cas9 protein (2 mg/mL) with
50 .mu.g 4-maleimidobutyryl-GGGRRRRRRRRRLLLL (m9R; 2 mg/mL) in PBS
(pH 7.4) followed by incubation on a rotator at room temperature
for 2 h. To remove unconjugated 9mR, the samples are dialyzed
against DPBS (pH 7.4) at 4.degree. C. for 24 h using 50 kDa
molecular weight cutoff membranes. Cas9-m9R protein is collected
from the dialysis membrane and the protein concentration is
determined using the Bradford assay (Biorad).
[0137] Synthetic lgRNA or a mixture of synthetic lgRNAs is
complexed with CPP: lgRNA (1 .mu.g) in 1 .mu.l of deionized water
is gently added to the C3G9R4LC peptide (9R) in lgRNA:peptide
weight ratios that range from 1:2.5 to 1:40 in 100 .mu.l of DPBS
(pH 7.4). This mixture is incubated at room temperature for 30 min
and diluted 10-fold using RNase-free deionized water.
[0138] 150 .mu.l Cas9-m9R (2 .mu.M) protein is mixed with 100 .mu.l
lgRNA:9R (10:50 .mu.g) complex and the resulting mixture is applied
to the cells for 4 h at 37.degree. C. Cells can also be treated
with Cas9-m9R and lgRNA:9R sequentially.
[0139] The antiviral assay is performed according to reported
procedures (Hu, W. et al. Proc Natl Acad Sci USA 2014, 110:
11461-11466; Lin, Su. et al. Molecular Therapy--Nucleic Acids,
2014, 3, e186). Delivery to cell lines is based either cationic
lipid or CPP based delivery of Cas9-lgRNA complexes instead of
plasmid transfection/transduction using gRNA/Cas9 expression
vectors.
EXAMPLES
[0140] The following examples further illustrate embodiments of the
disclosed invention, which are not limited by these examples.
Example 1: Compound 4
##STR00030##
[0142] Compound 2 is prepared essentially according to a reported
procedure in 4 steps (Santner, T. et al. Bioconjugate Chem. 2014,
25, 188-195). Compound 1 is treated with 2-azidoethanol in
dimethylacetamide at 120.degree. C. at the presence of
BF.sub.3.OEt.sub.2, and the resulting 2'-azido nucleoside is
tritylated (DMTrCl, in pyridine, RT), and attached to an
amino-functionalized support.
[0143] To compound 2 (0.99 mmol) in THF/H.sub.2O (2:1) (18 mL) is
then added trimethylphosphine (1.5 mL, 1.5 mmol). Reaction is
shaken at room temperature for 8 h and washed thoroughly with
THF/H.sub.2O (2:1). Dioxane/H.sub.2O (1:1) (20 mL) and NaHCO.sub.3
(185 mg, 2.2 mmol) are added. The reaction mixture is cooled down
to 0.degree. C., and Fmoc-OSu (415 mg, 1.23 mmol) in dioxane (2 mL)
is added. The reaction is shaked for 15 min at 0.degree. C., and
then washed with water and then THF (3.times.20 mL) to give
compound 4.
Example 2: Compound 7
##STR00031##
[0145] 6-Benzoyl-5'-O-DMTr-adenosine 5 (1.24 g, 1.84 mmole) is
dissolved in THF (40 mL) and sodium hydride (60% dispersion in
mineral oil, 0.184 g, 4.6 mmole) is added in portions at 0.degree.
C. The reaction mixture is warmed up to room temperature and
stirred for 15 min. The reaction is then cooled to 0.degree. C.,
and propargyl bromide (80% in toluene, 0.44 mL, 3.98 mmole) is
added. The reaction is then stirred under reflux for 12 h.
Saturated aqueous sodium bicarbonate (10 mL) is added, and
volatiles are removed in vacuo. The resulting residue is dissolved
in DCM, and washed with water and saturated brine. The organic
layer is collected, dried over anhydrous sodium sulfate, and
concentrated till dryness in vacuo. The resulting residue is
purified by silica-gel chromatography (eluent: 97:3, DCM:MeOH, 0.5%
pyridine) to provide compound 6.
[0146] To a solution of compound 6 (0.30 g, 0.42 mmol) in anhydrous
dichloromethane (5 mL) and diisopropylethylamine (0.15 mL, 0.83
mmol), under nitrogen, is added
2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite (0.13 mL, 0.58
mmol) dropwise. The reaction is stirred at room temperature for 3
h, and then diluted with dichloromethane (25 mL). The resulting
solution is washed with saturated aqueous KCl (25 mL), dried from
anhydrous Na.sub.2SO.sub.4, and concentrated in vacuo. The residue
is purified by column chromatography (60% EtOAc/hexane, 0.5%
pyridine) to give compound 7.
Example 3: ON-11
##STR00032##
[0148] ON-11 is prepared using 2'-TBS protected RNA phosphoramidite
monomers with t-butylphenoxyacetyl protection of the A, G and C
nucleobases and unprotected uracil. 0.3 M Benzylthiotetrazole in
acetonitrile (Link Technologies) is used as the coupling agent,
t-butylphenoxyacetic anhydride as the capping agent and 0.1 M
iodine as the oxidizing agent. Oligonucleotide synthesis is carried
out on an Applied Biosystems 394 automated DNA/RNA synthesizer
using the standard 1.0 .mu.mole RNA phosphoramidite cycle. Compound
4 (20 mg) is packed into a twist column. All .beta.-cyanoethyl
phosphoramidite monomers are dissolved in anhydrous acetonitrile to
a concentration of 0.1 M immediately prior to use. Stepwise
coupling efficiencies are determined by automated trityl cation
conductivity monitoring and in all cases are >96.5%.
[0149] Fmoc is then cleaved by treatment with 20% piperidine in
DMF. The resulting 3'-end aminoethyl oligonucleotide is then
treated with NHS ester of 6-azido caproic acid in DMF.
[0150] Cleavage of oligonucleotides from the solid support and
deprotection are achieved by exposure to concentrated aqueous
ammonia/ethanol (3/1 v/v) for 2 h at room temperature followed by
heating in a sealed tube for 45 min at 55.degree. C. and
desilylation in 1.0 M TBAF in THF for 24 h.
[0151] The above resulting oligonucleotide is dissolved in 0.5 M
Na.sub.2CO.sub.3/NaHCO.sub.3 buffer (pH 8.75) and incubated with
succinimidyl-6-azidohexanate (20 eq.) in DMSO to give ON-11.
[0152] Purification of the oligonucleotide is carried out by
reversed-phase HPLC on a Gilson system using an XBridge.TM. BEH300
Prep C18 10 .mu.M 10.times.250 mm column (Waters) with a gradient
of acetonitrile in ammonium acetate (0% to 50% buffer B over 30
min, flow rate 4 mL/min), buffer A: 0.1 M ammonium acetate, pH 7.0,
buffer B: 0.1 M ammonium acetate, pH 7.0, with 50% acetonitrile.
Elution is monitored by UV absorption at 295 nm. After HPLC
purification, the oligonucleotide is desalted using an NAP-10
column.
##STR00033##
Example 4: ON-12
[0153] ON-12 is synthesized in a way similar to the synthesis of
ON-11, except that commercially available solid phase supported
uridine phosphoramidite is used, and the last nucleoside
phosphoramidite is compound 7.
[0154] The oligonucleotide is cleaved off the solid phase and fully
deprotected, and purified as in example 3.
Example 5: ON-13
##STR00034##
[0156] A solution of alkyne ON-12 and azide ON-11 (0.2 nmol of
each) in 0.2 M NaCl (50 .mu.L) is annealed for 30 min at room
temperature. In the meantime tris-hydroxypropyl triazole ligand (28
nmol in 42 .mu.L 0.2 M NaCl), sodium ascorbate (40 nmol in 4 .mu.L
0.2 M NaCl) and CuSO.sub.4.5H.sub.2O (4 nmol in 4.0 .mu.L 0.2 M
NaCl), are added under argon. The reaction mixture is kept under
argon at room temperature for the desired time, and formamide (50
.mu.L) is added. The reaction is analyzed by and loading directly
onto a 20% polyacrylamide electrophoresis gel, and purified by
reversed-phase HPLC.
Example 6: ON-17
[0157] The ON-14 oligonucleotide carrying a maleimido group is
incubated with 5'-SH-oligonucleotide (ON-15, 1:1 molar ratio) in
0.1 M triethylammonium acetate (TEAA) at pH 7.0 overnight at room
temperature. The solution is evaporated to dryness, and to the
resulting mixture is added ON-16 in 250 .mu.M final concentration
in phosphate-buffered saline (PBS), pH 7.4, containing 0.7% DMF and
incubated at room temperature. The reaction mixture is analyzed and
separated by HPLC to give ON-17.
Example 7: Cas9::ON-13 Complex
[0158] Cas9 protein: Recombinant Cas9 protein is available from New
England BioLabs, Inc. and other providers or purified from E. coli
by a routinely used protocol (Anders, C. and Jinek, M. Methods
Enzymol. 2014, 546, 1-20). The purity and concentration of Cas9
protein are analyzed by SDS-PAGE.
[0159] Cas9-lgRNA Complex:
[0160] Cas9 and lgRNA are preincubated in a 1:1 molar ratio in the
cleavage buffer to reconstitute the Cas9-lgRNA complex.
Sequence CWU 1
1
57194RNAArtificial SequencelgRNA, nn is either base-paired or not,
and joined by non-nucleotide linker between the two, n32 (A) and
n33 (B).misc_feature(1)..(20)n is a, c, g, or
umisc_feature(32)..(33)n is a, c, g, or u 1nnnnnnnnnn nnnnnnnnnn
guuuuagagc unnagcaagu uaaaauaagg cuaguccguu 60aucaacuuga aaaaguggca
ccgagucggu gcuu 94262RNAArtificial SequencetracrgRNA 5'-alkynyl
tracrRNA, synthetic oligonucleotides 2uagcaaguua aaauaaggcu
aguccguuau caacuugaaa aaguggcacc gagucggugc 60uu 62332RNAArtificial
SequencecrgRNA 3'-azido crRNA, synthetic
oligonucleotidesmisc_feature(1)..(20)n is a, c, g, or u 3nnnnnnnnnn
nnnnnnnnnn guuuuagagc ua 32494RNAArtificial SequencelgRNA crRNA and
tracrRNA are fused as a single whole RNA by nNt-linker between a32
and u33.misc_feature(1)..(20)n is a, c, g, or u 4nnnnnnnnnn
nnnnnnnnnn guuuuagagc uauagcaagu uaaaauaagg cuaguccguu 60aucaacuuga
aaaaguggca ccgagucggu gcuu 94542RNAStreptococcus
pyogenesmisc_feature(1)..(20)n is a, c, g, or u 5nnnnnnnnnn
nnnnnnnnnn guuuuagagc uaugcuguuu ug 42681RNAStreptococcus pyogenes
6ggaaccauuc aaaacagcau agcaaguuaa aauaaggcua guccguuauc aacuugaaaa
60aguggcaccg agucggugcu u 81794RNAArtificial SequencelgRNA formed
by two ligations between nn of crgRNA and tracrgRNA, and between
a71 and a72 in tracrgRNA.misc_feature(1)..(20)n is a, c, g, or
umisc_feature(32)..(33)n is a, c, g, or u 7nnnnnnnnnn nnnnnnnnnn
guuuuagagc unnagcaagu uaaaauaagg cuaguccguu 60aucaacuuga aaaaguggca
ccgagucggu gcuu 94894RNAArtificial SequencelgRNA formed by two
ligations between nn of crgRNA and tracrgRNA, and between u62 and
c63 in tracrgRNA.misc_feature(1)..(20)n is a, c, g, or
umisc_feature(32)..(33)n is a, c, g, or u 8nnnnnnnnnn nnnnnnnnnn
guuuuagagc unnagcaagu uaaaauaagg cuaguccguu 60aucaacuuga aaaaguggca
ccgagucggu gcuu 94994RNAArtificial SequencelgRNA formed by three
ligations between nn of crgRNA and tracrgRNA, between a71 and a72,
and between g85 and u86 in
tracrgRNA.Modified(1)..(20)misc_feature(1)..(20)n is a, c, g, or
umisc_feature(32)..(33)n is a, c, g, or u 9nnnnnnnnnn nnnnnnnnnn
guuuuagagc unnagcaagu uaaaauaagg cuaguccguu 60aucaacuuga aaaaguggca
ccgagucggu gcuu 941074RNAArtificial SequenceTruncated
lgRNAmisc_feature(1)..(20)n is a, c, g, or umisc_feature(32)..(33)n
is a, c, g, or u 10nnnnnnnnnn nnnnnnnnnn guuuuagagc unnagcaagu
uaaaaggcua guccguuauc 60aacuugaaaa agug 741178RNAArtificial
SequenceTruncated lgRNAmisc_feature(1)..(20)n is a, c, g, or
umisc_feature(30)..(31)n is a, c, g, or u 11nnnnnnnnnn nnnnnnnnnn
guuuuagagn ncaaguuaaa auaaggcuag uccguuauca 60auggcaccga gucggugc
781275RNAArtificial SequenceTruncated lgRNAmisc_feature(1)..(20)n
is a, c, g, or umisc_feature(31)..(32)n is a, c, g, or u
12nnnnnnnnnn nnnnnnnnnn guuuuagagc nnguuaaaau aaggcuaguc cguuaucaau
60ggaccgaguc ggugc 751374RNAArtificial SequenceTruncated
lgRNAmisc_feature(1)..(20)n is a, c, g, or umisc_feature(29)..(30)n
is a, c, g, or u 13nnnnnnnnnn nnnnnnnnnn guuuuagcnn guuaaaauaa
ggcuaguccg uuaucaaugg 60caccgagucg gugc 741473RNAArtificial
SequenceTruncated lgRNAmisc_feature(1)..(20)n is a, c, g, or
umisc_feature(31)..(32)n is a, c, g, or u 14nnnnnnnnnn nnnnnnnnnn
guuuuagagc nnguuaaaau aaggcuaguc cguaaauggc 60accgagucgg ugc
731567RNAArtificial SequenceTruncated lgRNAmisc_feature(1)..(20)n
is a, c, g, or umisc_feature(29)..(30)n is a, c, g, or u
15nnnnnnnnnn nnnnnnnnnn guuuuagcnn guuaaaaggc uaguccguaa uggcaccgag
60ucggugc 671630RNAArtificial SequenceON-1 3'-amino crgRNA.
16gcguggccug ggcgggacug guuuuagaga 301730RNAArtificial SequenceON-2
3'-azido crgRNA. 17gcguggccug ggcgggacug guuuuagaga
301861RNAArtificial SequenceON-3 solid supported tracrgRNA after
detritylation. 18agcaaguuaa aauaaggcua guccguuauc aacuugaaaa
aguggcaccg agucggugcu 60u 611961RNAArtificial SequenceON-4 solid
supported 5'-alkynyl tracrgRNA before deprotection 19agcaaguuaa
aauaaggcua guccguuauc aacuugaaaa aguggcaccg agucggugcu 60u
612061RNAArtificial SequenceON-5 5'-alkynyl tracrgRNA. 20agcaaguuaa
aauaaggcua guccguuauc aacuugaaaa aguggcaccg agucggugcu 60u
612161RNAArtificial SequenceON-6 solid supported 5'-alkynyl
(strained) tracrgRNA before deprotection. 21agcaaguuaa aauaaggcua
guccguuauc aacuugaaaa aguggcaccg agucggugcu 60u 612261RNAArtificial
SequenceON-7 5'-alkynyl (strained) tracrgRNA. 22agcaaguuaa
aauaaggcua guccguuauc aacuugaaaa aguggcaccg agucggugcu 60u
612361RNAArtificial SequenceON-8 solid supported 5'-thiol tracrgRNA
before deprotection. 23agcaaguuaa aauaaggcua guccguuauc aacuugaaaa
aguggcaccg agucggugcu 60u 612461RNAArtificial SequenceON-9 5'-thiol
tracrgRNA. 24agcaaguuaa aauaaggcua guccguuauc aacuugaaaa aguggcaccg
agucggugcu 60u 612530RNAArtificial SequenceON-10 3'-maleimidyl
crgRNA 25gcguggccug ggcgggacug guuuuagagc 302631RNAArtificial
SequenceON-11 3'-azido crgRNA. 26gcguggccug ggcgggacug guuuuagagc u
312761RNAArtificial SequenceON-12 5'-alkynyl tracrgRNA.
27agcaaguuaa aauaaggcua guccguuauc aacuugaaaa aguggcaccg agucggugcu
60u 612892RNAArtificial SequenceON-13 lgRNA ligated between 31u and
32a. 28gcguggccug ggcgggacug guuuuagagc uagcaaguua aaauaaggcu
aguccguuau 60caacuugaaa aaguggcacc gagucggugc uu
922931RNAArtificial SequenceON-14 3'-maleimidyl crgRNA 29gcguggccug
ggcgggacug guuuuagagc a 313029RNAArtificial SequenceON-15 5'-thiol
3'-azido tracrgRNA1 30ugcaaguuaa aauaaggcua guccguuau
293132RNAArtificial SequenceON-16 5'-alkynyl tracrgRNA2.
31caacuugaaa aaguggcacc gagucggugc uu 323292RNAArtificial
SequenceON-17 lgRNA ligated between a31 and u32, and between u60
and c61. 32gcguggccug ggcgggacug guuuuagagc augcaaguua aaauaaggcu
aguccguuau 60caacuugaaa aaguggcacc gagucggugc uu
923359RNAArtificial SequenceON-18 3'-amino ligated crgRNA and
tracrgRNA1. 33gcguggccug ggcgggacug guuuuagagc ugcaaguuaa
aauaaggcua guccguuau 593459RNAArtificial SequenceON-19 3'-azido
ligated crgRNA and tracrgRNA1. 34gcguggccug ggcgggacug guuuuagagc
ugcaaguuaa aauaaggcua guccguuau 593591RNAArtificial SequenceON-20
lgRNA. 35gcguggccug ggcgggacug guuuuagagc ugcaaguuaa aauaaggcua
guccguuauc 60aacuugaaaa aguggcaccg agucggugcu u 913623DNAHBV
36ctctgctaga tcccagagtg agg 233723DNAHBV 37gctatcgctg gatgtgtctg
cgg 233825DNAHBV 38tggacttctc tcaattttct agggg 253923DNAHBV
39gggggatcac ccgtgtgtct tgg 234025DNAHBV 40tatgtggatg atgtggtact
ggggg 254121DNAHBV 41cctcaccata cagcactcgg g 214226DNAHBV
42gtgttggggt gagttgatga atctgg 264325DNAHIV 43tatgtggatg atgtggtact
ggggg 254425DNAHIV 44tatatggatg atgtggtatt ggggg 254525DNAHIV
45tatgtggatg atgtggtatt ggggg 254625DNAHIV 46tatatagatg atgtggtact
ggggg 254723DNAHIV 47gattggcaga actacacacc agg 234823DNAHIV
48atcagatatc cactgacctt tgg 234923DNAHIV 49gcgtggcctg ggcgggactg
ggg 235023DNAHIV 50cagcagttct tgaagtactc cgg 235123DNAEBV
51gccctggacc aacccggccc ggg 235223DNAEBV 52ggccgctgcc ccgctccggg
tgg 235322DNAEBV 53ggaagacaat gtgccgccat gg 225423DNAEBV
54tctggaccag aaggctccgg cgg 235523DNAEBV 55gctgccgcgg agggtgatga
cgg 235623DNAEBV 56ggtggcccac cgggtccgct ggg 235723DNAEBV
57gtcctcgagg gggccgtcgc ggg 23
* * * * *