U.S. patent application number 12/581871 was filed with the patent office on 2010-07-22 for safe lentiviral vectors for targeted delivery of multiple therapeutic molecules.
Invention is credited to Jeffrey A. Galvin, Zhennan Lai.
Application Number | 20100183558 12/581871 |
Document ID | / |
Family ID | 42106949 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100183558 |
Kind Code |
A1 |
Lai; Zhennan ; et
al. |
July 22, 2010 |
SAFE LENTIVIRAL VECTORS FOR TARGETED DELIVERY OF MULTIPLE
THERAPEUTIC MOLECULES
Abstract
The present application discloses a lentiviral transfer system
which includes: (i) a self-inactivating transfer vector comprising:
multiple gene units, wherein each gene unit includes a heterologous
nucleic acid sequence operably linked to a regulatory nucleic acid
sequence; and (ii) a helper construct which lacks a 5' LTR, wherein
the 5' LTR has been replaced with a heterologous promoter, in which
the helper construct further comprises: a lentiviral env nucleic
acid sequence containing a deletion, wherein the deleted env
nucleic acid sequence does not produce functional env protein; and
a packaging signal contains a deletion, wherein the deleted
packaging signal is nonfunctional.
Inventors: |
Lai; Zhennan; (North
Potomac, MD) ; Galvin; Jeffrey A.; (Redwood City,
CA) |
Correspondence
Address: |
JHK Law
P.O. Box 1078
La Canada
CA
91012-1078
US
|
Family ID: |
42106949 |
Appl. No.: |
12/581871 |
Filed: |
October 19, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61243121 |
Sep 16, 2009 |
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61116138 |
Nov 19, 2008 |
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61196457 |
Oct 17, 2008 |
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Current U.S.
Class: |
424/93.2 ;
435/320.1 |
Current CPC
Class: |
C12N 2830/008 20130101;
C07K 14/4746 20130101; C12N 2320/32 20130101; A61K 31/7088
20130101; C12N 15/86 20130101; C12N 2310/14 20130101; C12N 2830/20
20130101; C07K 2319/10 20130101; C12N 15/1135 20130101; A61K 48/00
20130101; A61P 3/00 20180101; A61P 43/00 20180101; C12N 2330/51
20130101; A61P 35/00 20180101; C12N 2740/16043 20130101 |
Class at
Publication: |
424/93.2 ;
435/320.1 |
International
Class: |
A61K 35/76 20060101
A61K035/76; A61P 35/00 20060101 A61P035/00; A61P 43/00 20060101
A61P043/00; C12N 15/63 20060101 C12N015/63 |
Claims
1. A lentiviral transfer system comprising: (i) a self-inactivating
transfer vector comprising: multiple gene units, wherein each gene
unit comprises a heterologous nucleic acid sequence operably linked
to a regulatory nucleic acid sequence; and (ii) a helper construct
which lacks a 5' LTR, wherein said 5' LTR has been replaced with a
heterologous promoter, said helper construct further comprising: a
lentiviral env nucleic acid sequence containing a deletion, wherein
said deleted env nucleic acid sequence does not produce functional
env protein; a packaging signal containing a deletion, wherein said
deleted packaging signal is nonfunctional.
2. The lentiviral transfer system of claim 1, wherein in the
transfer vector, one or more of said regulatory nucleic acid
sequences comprises a general promoter.
3. The lentiviral transfer system of claim 2, wherein said general
promoter is CMV-IE promoter.
4. The lentiviral transfer system of claim 1, wherein in the
transfer vector, one or more of said regulatory nucleic acid
sequences comprises a cell or tissue-specific promoter.
5. The lentiviral transfer system of claim 4, wherein said cell or
tissue-specific promoter is selected from the group consisting of:
TSTA promoter, mesothelin promoter, hPSA promoter, hCCKAR promoter,
hAFP promoter, and hNSE promoter.
6. The lentiviral transfer system of claim 1, wherein in the
transfer vector, a translation initiation site is located between
gene units.
7. The lentiviral transfer system of claim 6, wherein the
translation initiation site is internal ribosome entry site
(IRES).
8. The lentiviral transfer system of claim 1, wherein the
heterologous nucleic acid sequence encodes an RNAi or a
polypeptide.
9. The lentiviral transfer system of claim 8, wherein the
polypeptide or RNAi inhibits expression or activity of a gene or
protein that contributes to progression of cancer.
10. The lentiviral transfer system of claim 8, wherein the
polypeptide or RNAi inhibits expression or activity of a tumor
promoting gene or protein.
11. The lentiviral transfer system of claim 8, wherein the
polypeptide or RNAi inhibits expression or activity of a growth
factor, growth factor receptor, angiogenic factor, angiogenic
factor receptor, cell cycle regulator, apoptosis-inducing molecule,
or cell adhesion molecule.
12. The lentiviral transfer system of claim 8 wherein the
polypeptide or RNAi inhibits the expression or activity of a
vascular endothelial growth factor, a vascular endothelial growth
factor receptor, epidermal growth factor receptor, hTR, hTERT,
papillomavirus E6, papillomavirus E7, BCR-abl, CEACAM6, MMP9, or a
cathepsin.
13. The lentiviral transfer system of claim 8, wherein said RNAi is
targeted to Bcl-2, AEC-1, Myc or K-ras.
14. The lentiviral transfer system of claim 8, wherein said
polypeptide is P53 protein.
15. The lentiviral transfer system of claim 8, wherein said
heterologous nucleic acid sequence encodes the P53 protein and RNAi
is targeted to Bcl-2.
16. The lentiviral transfer system of claim 1, wherein in the
transfer vector, a sequence encoding a cell or tissue-specific
enzyme cleavage site is located between one or more gene units,
wherein cleavage at the site occurs within a fused polypeptide that
is expressed by the heterologous nucleic acid sequences of the gene
units.
17. The lentiviral transfer system of claim 16, wherein said cell
or tissue-specific enzyme cleavage site is a protease 2A cleavage
site, a presecretory protein signal peptidase cleavage site, or a
pancreatic prechymotrypsinogen cleavage site.
18. The lentiviral transfer system of claim 16, wherein one of the
gene units is an intercellular trafficking signal.
19. The lentiviral transfer system of claim 18 wherein the
intercellular trafficking signal is a membrane-penetrating protein
or a fragment thereof.
20. The lentiviral transfer system of claim 18 wherein the
membrane-penetrating protein is a plant or bacterial protein
toxin.
21. The lentiviral transfer system of claim 18 wherein the
membrane-penetrating protein is a viral protein.
22. The lentiviral transfer system of claim 21, wherein said
trafficking signal is derived from herpesvirus VP22 or HIV-Tat.
23. The lentiviral transfer system of claim 22, wherein said
trafficking signal is the HIV-Tat eleven amino acid transduction
sequence.
24. The lentiviral transfer system of claim 21, wherein said
herpesvirus is HSV1.
25. The lentiviral transfer system of claim 21, wherein said
trafficking signal is a VP22 protein homologue of HSV1 VP22.
26. The lentiviral transfer system of claim 25 wherein the VP22
transport signal comprises the C-terminal 34 amino acid sequence of
VP22 of HSV1, or a fragment having 80% or greater identity to the
terminal 34 amino acid sequence of VP22 of HSV1.
27. The lentiviral transfer system of claim 22 wherein the VP22
transport signal comprises one or more of RSASR, RTASR, RSRAR,
RTRAR, ATATR, or RSAASR.
28. The lentiviral transfer system of claim 1, wherein expression
of the heterologous nucleic acid sequences in the transfer vector
inhibits progression of a disease or disorder.
29. The lentiviral transfer system of claim 28, wherein expression
of the multiple heterologous nucleic acid sequences synergistically
inhibits progression of a disease or disorder.
30. The lentiviral transfer system of claim 28, wherein said
disease or disorder is cancer.
31. The lentiviral transfer system of claim 28, wherein said
disease or disorder is a genetic disorder.
32. The lentiviral transfer system of claim 31, wherein said
genetic disorder is metabolic disorder.
33. The lentiviral transfer system of claim 32, wherein said
metabolic disorder is Gaucher's Disease or Fabry's Disease.
34. The lentiviral transfer system of claim 28, wherein said
disease or disorder is a neurological disorder.
35. The lentiviral transfer system of claim 34, wherein said
neurological disorder is Alzheimer's Disease or Parkinson's
Disease.
36. A method for treating a condition, comprising administering to
a patient a lentiviral particle for gene transfer, said lentiviral
particle produced using a lentiviral transfer system comprising:
(i) a transfer vector comprising: multiple gene units, wherein each
gene unit comprises a heterologous nucleic acid sequence operably
linked to a regulatory nucleic acid sequence; and (ii) a helper
construct which lacks a 5' LTR, wherein said 5' LTR has been
replaced with a heterologous promoter, said helper construct
further comprising: a lentiviral env nucleic acid sequence
containing a deletion, wherein said deleted env nucleic acid
sequence does not produce functional env protein; a packaging
signal containing a deletion, wherein said deleted packaging signal
is nonfunctional.
37. The method of claim 36, wherein said condition is a cancer.
38. The method of claim 37, wherein said cancer is liver cancer,
pancreatic cancer, or prostate cancer.
39. The method according to claim 37, wherein the cancer is
prostate cancer, and wherein the heterologous nucleic acid sequence
encodes a P53 protein and an RNAi targeted for Bcl-2.
40. The method of claim 36, wherein said condition is a genetic
disorder.
41. The method of claim 36, wherein said condition is a need for
cosmetic enhancement.
42. A pharmaceutical composition comprising a lentiviral particle
for gene transfer, said lentiviral particle produced using a
lentiviral transfer system comprising: (i) a self-inactivating
transfer vector comprising: multiple gene units, wherein each gene
unit comprises a heterologous nucleic acid sequence operably linked
to a regulatory nucleic acid sequence; and (ii) a helper construct
which lacks a 5' LTR, wherein said 5' LTR has been replaced with a
heterologous promoter, said helper construct further comprising: a
lentiviral env nucleic acid sequence containing a deletion, wherein
said deleted env nucleic acid sequence does not produce functional
env protein; a packaging signal containing a deletion, wherein said
deleted packaging signal is nonfunctional.
43. The pharmaceutical composition according to claim 42, wherein
the heterologous nucleic acid sequence encodes a P53 protein and an
RNAi targeted for Bcl-2, which expressed both respectively or at
the same time.
44. The pharmaceutical composition of claim 42, further comprising
a chemotherapeutic agent or a steroid agent.
45. The pharmaceutical composition of claim 44, wherein the steroid
agent is prednisolone, cortisone, corticosterone, or
dexamethasone.
46. A pharmaceutical composition comprising a self inactivating
lentiviral transfer vector comprising multiple gene units, wherein
each gene unit comprises a heterologous nucleic acid sequence
operably linked to a regulatory nucleic acid sequence.
47. The pharmaceutical composition according to claim 46, wherein
the heterologous nucleic acid sequence encodes a P53 protein and an
RNAi targeted for Bcl-2.
48. The lentiviral transfer system of claim 1, wherein the transfer
vector further comprises mammalian insulator sequence and splice
acceptor and splice donor sites, and is free of wPRE (wood-chuck
hepatitis virus post-transcriptional element) downstream of a
cloning site.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of priority to
U.S. Provisional Application No. 61/243,121, filed Sep. 16, 2009;
U.S. Provisional Application No. 61/116,138, filed Nov. 19, 2008;
and U.S. Provisional Application No. 61/196,457, filed Oct. 17,
2008, the contents of which are incorporated by reference herein in
their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of medicine,
specifically to delivery of multiple therapeutic molecules
strategically combined with regulatory elements, using a safe for
human use, highly and long-term expressing in human subject
lentiviral gene transfer vector for the treatment of a condition,
disease or disorder.
[0004] 2. General Background and State of the Art
[0005] The present application relates to a gene transfer vector
that provides versatility, control, high expression, stable
multiple gene expression, tolerability, and safety, for the design
of effective gene therapies for disease. Virus vectors in
development each have limitations for researchers to address.
[0006] Identification of target genes involved in neoplastic
transformation and tumor progression has encouraged the idea that
nucleotide sequences of cancer-relevant genes could lead to the
development of tailored anticancer agents that lack many of the
toxic side effects of traditional cytotoxic drugs. Mutations of the
p53 gene are associated with transformation to a malignant
phenotype. Transfer of wild-type P53, which plays a critical role
in the regulation of cell growth and downregulation of genes that
contribute to cancer progression, is hoped to result in selective
and specific inhibition of tumor growth while minimizing
undesirable side effects on normal cells. Delivery of the p53 gene
has been reported in a replication-deficient adenoviral vector
containing the wild-type p53 gene sequence. Functional activity and
expression of the transgene product in tumor cells treated with the
adenoviral vector has been reported. (Baker, et al., 1990,
Suppression of human colorectal carcinoma cell growth by wild-type
p53, Science 249: 912-915.) Two adenovirus-based gene therapeutics
for the treatment of cancer were recently commercialized in China.
These two agents, combined with chemotherapy, have been used there
as an alternative treatment for some types of refractory cancer.
(Peng, 2005, Current Status of Gendicine in China: Recombinant
Human Ad-p53 Agent for Treatment of Cancers, Hum. Gene Ther. 16,
1016-1027; Yu, W., and Fang, H., 2007, Clinical Trials with
Oncolytic Adenovirus in China. Curr. Cancer Drug Targets 7,
141-148). Although adenovirus vectors can efficiently deliver
therapeutic genes in both dividing and non-dividing cells, and can
be manufactured at high viral titers, adenovirus vectors are highly
immunogenic (Shirakawa, et al., 2008, The Current Status of
Adenovirus-based Cancer Gene Therapy, Mol. Cells. 25(4): 462-466).
Furthermore, long-term expression in target tissues is not
observed.
[0007] Lentiviruses, such as HIV, are "slow viruses." Vectors
derived from lentiviruses can be expressed long-term in the host
cells after a few administrations to the patients, e.g., via ex
vivo transduced bone marrow stem cells. For most diseases and
disorders, including genetic diseases, cancer, and neurological
disease, long-term expression is crucial to successful treatment.
Safety has been a concern with lentiviral vectors, but a number of
strategies for eliminating the ability of lentiviral vectors to
replicate have now been described. For example, the deletion of
promoter and enhancer elements from the U3 region of the long
terminal repeat (LTR) are thought to have no LTR-directed
transcription. The resulting vectors are called "self-inactivating"
(SIN). However, it has been reported that HIV-1-derived vectors
containing the SIN deletion in the U3 region of the LTR are capable
of expressing full-length genomic transcripts (Logan, et al., 2004,
Integrated Self-Inactivating Lentiviral Vectors Produce Full-Length
Genomic Transcripts Competent for Encapsidation and Integration, J.
Virology 78(16): 8421-8436). Therefore, combination of this
deletion with other safety measures must be considered.
[0008] The last few years have seen immense excitement regarding
the use of RNAi agents for disease therapies. Investigators
reported that they were able to specifically silence mutant
oncogenic ras without affecting wild-type ras in vitro (Zhang, et
al., 1995, Safety evaluation of Ad5CMV-p53 in vitro and in vivo,
Human Gene Therapy 6:155-164; Ishii, et al., 2001, Potential cancer
therapy with the fragile histidine triad gene review of the
preclinical studies, JAMA 286: 2441-2449). It is believed that
treatment costs for siRNA would be similar to most protein-based
therapies, e.g., antibody therapies. Preclinical cancer studies
have shown inhibition of growth and survival of tumor cells by
RNAi-mediated downregulation of several key oncogenes or
tumor-promoting genes, including growth and angiogenic factors or
their receptors (vascular endothelial growth factor, epidermal
growth factor receptor), human telomerase (hTR, hTERT), viral
oncogenes (papillomavirus E6 and E7) or translocated oncogenes
(BCR-abl).
[0009] Various studies report on the in vivo activity and the
potential of RNAi agents to suppress tumor growth. These include an
intratumoral injection of an shRNA-adenoviral vector construct
targeting a cell-cycle regulator causing inhibition of subcutaneous
small cell lung tumor in mice, and systemic administration of an
siRNA targeting a carcinoembryonic antigen-related cell adhesion
molecule (CEACAM6) in mice with subcutaneously xenografted
pancreatic adenocarcinoma cells. In another report, direct
injection of a plasmid vector expressing shRNAs to matrix
metalloproteinase MMP-9 and a cathepsin showed efficacy in
established glioblastoma (Chen, et al., 2005, Reversal of the
phenotype by K-rasval12 silencing mediated by adenovirus-delivered
siRNA in human pancreatic cancer cell line Panc-1, World J.
Gastroenterol. 11(6): 831-838). However, delivery of siRNA for
long-term expression in target cells and tissues has been
particularly difficult in vivo.
[0010] Another problem in gene transfer is the delivery of
therapeutic molecules to a sufficient number of target cells to
elicit a therapeutic response. Recently, a series of virus-encoded
and other regulatory proteins were found to possess the ability to
cross biological membranes. These proteins include HIV-Tat and the
herpes simplex virus type 1 tegument protein VP22. VP22 was also
reported to exhibit a unique property of effecting intercellular
spread. VP22 is a basic, 38-kDa phosphorylated protein (Knopf, et
al., 1980, J. Gen. Virol. 46:405-414) encoded by the viral UL49
gene (Elliott, et al., 1992, J. Gen. Virol. 73:723-726).
[0011] Specific and controlled delivery of therapeutic molecules to
an affected cell population, e.g., to tumor cells and even
circulating cancer cells, can potentially be achieved by
strategically positioning nucleic acid and protein regulatory
elements, e.g., cell and tissue-specific promoters and enzyme
cleavage sites. These elements are recognized by the production
machinery that is present only in certain cell types. The ability
to easily combine and regulate the expression and delivery of
multiple therapeutic molecules, while taking effective safety
measures without compromising expression levels, in methods for
using a lentiviral gene transfer vector or lentiviral transfer
system, would provide a researcher with a critical tool for
treating a broad range of diseases and disorders.
SUMMARY OF THE INVENTION
[0012] The present invention is related to lentiviral transfer
systems including safe, self-inactivating, recombinant lentiviral
vectors with the capacity to accommodate strategic combinations of
genes for therapeutic molecules and novel regulatory sequences, in
methods for treating a broad range of diseases and disorders.
[0013] In one aspect, the invention relates to a lentiviral gene
transfer system comprising: a self-inactivating transfer vector
comprising: a first gene unit with a first heterologous nucleic
acid sequence, operably linked to a first regulatory nucleic acid
sequence; and a second gene unit with a second heterologous nucleic
acid sequence, operably linked to a second regulatory nucleic acid
sequence; and a helper construct which lacks a 5' LTR, wherein said
5' LTR has been replaced with a heterologous promoter, said helper
construct further comprising: a lentiviral env nucleic acid
sequence containing a deletion, wherein said deleted env nucleic
acid sequence does not produce a functional env protein; a
packaging signal containing a deletion, wherein said deleted
packaging signal is nonfunctional. The transfer vector may be
preferably derived from HIV-1. Preferably, RNAi or a polypeptide
may be encoded by the heterologous nucleic acid sequence. In
addition, expression of the first and second heterologous nucleic
acid sequences may have a synergistic effect in inhibiting
progression of a disease or disorder.
[0014] The transfer vector may include mammalian insulator sequence
and splice acceptor and splice donor sites, and may be free of wPRE
"wood-chuck" hepatitis virus post-transcriptional element
downstream of a cloning site (Gao et al., J. Virol., March 2008; p.
2938-2951).
[0015] The RNAi may inhibit expression of a gene that contributes
to progression of a disease or disorder. In addition, the first or
second heterologous nucleic acid sequence may include a sequence
encoding a trafficking signal, and the trafficking signal may be
expressed as a fusion with a protein expressed from the second
heterologous nucleic acid sequence. The intercellular trafficking
signal may be a membrane-penetrating protein or a fragment thereof,
such as a plant or bacterial protein toxin, or viral protein, any
other sequence domain with transporting function between cells, in
particular, cancer cells. The trafficking signal may be derived
from herpesvirus VP22 or HIV-Tat, or may be a HIV-Tat eleven amino
acid transduction sequence. The herpesvirus may be HSV1, and the
trafficking signal may further be a VP22 protein homologue of HSV1
VP22. The VP22 transport signal may include a C-terminal 34 amino
acid sequence of VP22 of HSV1, or a fragment having 80% or greater
identity to the terminal 34 amino acid sequence of VP22 of HSV1.
Further, the VP22 transport signal may include one or more of
RSASR, RTASR, RSRAR, RTRAR, ATATR, or RSAASR.
[0016] The transfer vector may utilize general or cell or tissue
specific promoters such as TSTA promoter, mesothelin promoter, hPSA
promoter, hCCKAR promoter, hAFP promoter, and hNSE promoter.
[0017] Tissue-specific enzyme cleavage sites may be included in the
transfer vector, wherein cleavage at the site occurs within a
polypeptide that is encoded by the first and second heterologous
nucleic acid sequences. The regulatory nucleic acid sequence may
include a sequence encoding a cell or tissue-specific enzyme
cleavage site, wherein cleavage at the site occurs within at least
one polypeptide that is encoded by two or more of the first, second
and third heterologous nucleic acid sequences. The cell or
tissue-specific enzyme cleavage site may be a protease 2A cleavage
site, a presecretory protein signal peptidase cleavage site, or a
pancreatic prechymotrypsinogen cleavage site.
[0018] The transfer vector may also include a nucleic acid sequence
encoding translation initiation site. Such a sequence may be
positioned between the gene units, and in certain aspects may be
considered to belong to a "regulatory sequence" of a gene unit.
Although a variety of sequences may be used, an internal ribosome
entry site (IRES) is preferred.
[0019] The inventive lentiviral transfer system may be used to
prepare a treatment for a disease or disorder. The disease or
disorder may be cancer. In a preferred embodiment, a heterologous
nucleic acid sequence may encode the P53 protein.
[0020] The antisense RNA, RNAi, or any polypeptide expressed from
the heterologous nucleic acid sequence may be expressed
consistently and for long period of time to inhibit expression of a
gene or the activity of a gene product that contributes to
progression of the cancer. The antisense RNA, RNAi and the
polypeptide may inhibit expression and activity of a tumor
promoting gene or gene product. The RNAi and the expressed
polypeptide may inhibit expression of or activity of a growth
factor, growth factor receptor, angiogenic factor, angiogenic
factor receptor, cell cycle regulator, apoptosis-inducing molecule,
or cell adhesion molecule. The RNAi or the expressed polypeptide
may inhibit the expression or activity of a vascular endothelial
growth factor, Bcl-2, K-ras, AEC-1, Myc, including c-Myc, a
vascular endothelial growth factor receptor, epidermal growth
factor receptor, hTR, hTERT, papillomavirus E6, papillomavirus E7,
BCR-abl, CEACAM6, MMP9, or a cathepsin.
[0021] The cancer to be treated may be prostate cancer in which a
regulatory nucleic acid sequence may include hPSA. If the cancer is
liver cancer then a regulatory nucleic acid sequence may include
hAFP. If the cancer is pancreatic cancer then a regulatory nucleic
acid sequence may include hCCKAR to control the expression of RNAi
or a polypeptide.
[0022] The inventive lentiviral transfer system may be used to
prepare a treatment for a genetic disorder, such as a metabolic
disorder, including Gaucher's Disease or Fabry's Disease. In the
case of Gaucher's Disease, a first heterologous nucleic acid
sequence may encode glucocerebrosidase and a second heterologous
nucleic acid sequence may encode a human intrinsic selectable
marker such as huCD25 protein or huNGF protein.
[0023] The lentiviral transfer system may include a regulatory
nucleic acid sequence that includes a sequence encoding a
trafficking signal that is expressed as a fusion with a
glucocerebrosidase protein expressed from an adjacent heterologous
nucleic acid sequence. A second regulatory nucleic acid sequence
may include translation initiation sequence as well. A cell or
tissue-specific enzyme cleavage site may also be included.
[0024] In the case of Fabry's Disease, said first heterologous
nucleic acid sequence may encode an alpha-galactosidase-A protein,
and a second heterologous nucleic acid sequence may encode the
huCD25 protein.
[0025] In another aspect, the inventive lentiviral transfer system
may include a first heterologous nucleic acid sequence comprising a
sequence encoding a trafficking signal that is expressed as a
fusion with the alpha-galactosidase-A protein expressed from the
first heterologous nucleic acid sequence. The trafficking signal
may be a VP22 trafficking signal or an HIV-Tat trafficking
signal.
[0026] In yet another aspect, if the genetic disorder is Leber
Congenital Amaurosis, the first heterologous nucleic acid sequence
may encode the RPE65 protein, and a second heterologous nucleic
acid sequence may encode the hBDNF protein, and a second regulatory
nucleic acid sequence may include hNSE, and further the vector may
include a third heterologous nucleic acid sequence encoding the
hNGF protein, and also a third regulatory nucleic acid sequence
including a sequence encoding a cell or tissue-specific enzyme
cleavage site.
[0027] In another aspect, the disease or disorder to be treated may
be a neurological disorder, such as Alzheimer's Disease, in which
case, a first heterologous nucleic acid may encode the hNGF
protein, a second heterologous nucleic acid may encode an RNAi
targeted to beta-amyloid precursor protein, and wherein the second
regulatory nucleic acid sequence may include a sequence encoding a
cell or tissue-specific enzyme cleavage site. A third heterologous
nucleic acid that encodes the hBDNF protein may also be included,
in which the first regulatory nucleic acid sequence may include a
sequence encoding a trafficking signal that is expressed as a
fusion with the hNGF protein expressed from the first heterologous
nucleic acid sequence.
[0028] If the neurological disorder is Parkinson's Disease, a first
heterologous nucleic acid may encode the hBDNF protein or the hGDNF
protein, and the second heterologous nucleic acid encodes hGAD. The
second regulatory nucleic acid sequence may include a sequence
encoding a cell or tissue-specific enzyme cleavage site. Further,
the first regulatory nucleic acid may include a sequence encoding a
trafficking signal that is expressed as a fusion with the hBDNF
protein or hGDNF protein expressed from the first heterologous
nucleic acid sequence, wherein the vector may further include a
third heterologous nucleic acid sequence encoding hNGF, in which
the vector may further include a third regulatory nucleic acid
sequence encoding a cell or tissue-specific enzyme cleavage
site.
[0029] In another aspect, the invention is directed to a method for
treating a condition, comprising administering to a patient a
lentiviral particle for gene transfer, said lentiviral particle
produced using a lentiviral transfer system comprising: a
self-inactivating transfer vector comprising: a first gene unit
with a first heterologous nucleic acid sequence, operably linked to
a first regulatory nucleic acid sequence; and a second gene unit
with a second heterologous nucleic acid sequence, operably linked
to a second regulatory nucleic acid sequence; and a helper
construct which lacks a 5' LTR, wherein said 5' LTR has been
replaced with a heterologous promoter, said helper construct
further comprising: a lentiviral env nucleic acid sequence
containing a deletion, wherein said deleted env nucleic acid
sequence does not produce functional env protein; a packaging
signal containing a deletion, wherein said deleted packaging signal
is nonfunctional. The condition may be cancer, such as liver
cancer, pancreatic cancer, or prostate cancer. The condition may
also be a genetic disorder, such as Gaucher's Disease or Fabry's
Disease. The condition may also be a neurological disorder, such as
Parkinson's Disease or Alzheimer's Disease. The condition may also
be a need for cosmetic enhancement.
[0030] A pharmaceutical composition comprising a lentiviral
particle for gene transfer, said lentiviral particle produced using
a lentiviral transfer system comprising: a self-inactivating
transfer vector comprising: a first gene unit with a first
heterologous nucleic acid sequence, operably linked to a first
regulatory nucleic acid sequence; and a second gene unit with a
second heterologous nucleic acid sequence, operably linked to a
second regulatory nucleic acid sequence; and a helper construct
which lacks a 5' LTR, wherein said 5' LTR has been replaced with a
heterologous promoter, said helper construct further comprising: a
lentiviral env nucleic acid sequence containing a deletion, wherein
said deleted env nucleic acid sequence does not produce functional
env protein; a packaging signal containing a deletion, wherein said
deleted packaging signal is nonfunctional.
[0031] In a further aspect, the invention is directed to a
pharmaceutical composition that includes a lentiviral transfer
vector, said lentiviral transfer vector comprising a first
heterologous nucleic acid sequence, operably linked to a first
regulatory nucleic acid sequence; and a second heterologous nucleic
acid sequence, operably linked to a second regulatory nucleic acid
sequence, wherein said transfer vector is self-inactivating.
[0032] The pharmaceutical compositions as described above may
further include a chemotherapeutic agent, a steroid agent such as
prednisolone, cortisone, corticosterone, or dexamethasone.
[0033] In one aspect of the invention, SIN element is incorporated
into the recombinant lentivirus; the tat region has been modified
so as to optionally allow for infection efficiency without allowing
the replication functions and uncontrolled infection of the
recombinant lentivirus beyond the intended target; and the rev
protein has been inactivated so as to prevent further unwanted
infectivity while preserving the basic function of the rev to
support the expression efficiency of the therapeutic gene(s). Tat
and rev proteins may be inactivated of their original replication
activity without necessarily removing them from the lentivirus and
thereby preserve their desirable attributes while keeping the
resulting vector bio-safe.
[0034] These and other objects of the invention will be more fully
understood from the following description of the invention, the
referenced drawings attached hereto and the claims appended
hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The present invention will become more fully understood from
the detailed description given herein below, and the accompanying
drawings which are given by way of illustration only, and thus are
not limitative of the present invention, and wherein;
[0036] FIG. 1 Lentiviral transfer system. The drawing shows an
HIV-1-based gene transfer system carrying multiple functional
(therapeutic) genes. A. Helper (packaging) construct. The 5' LTR of
the helper construct is replaced with the CMV promoter, to avoid
integration of the viral elements presented in that construct. This
is an important biosafety feature of the lentiviral transfer system
of the invention. The triangles represent deletions: one represents
a 36-bp deletion harboring the putative packaging signal from
nucleotides 753 to 789 between the 5' major splice donor site and
the beginning of the gag ATG coding region; one represents a
deletion in the tat gene, and a third represents a deletion in nef.
The packaging signal is functionally absent from this construct, to
avoid production of an active gag-pol precursor. The poly (A) site
was derived from the bovine growth hormone gene. The helper
construct provides a nucleic acid sequence encoding lentiviral gag
and pol, operably linked to a heterologous regulatory nucleic acid
sequence. The construct further contains a deleted, nonfunctional
env protein and is devoid of lentiviral sequences both upstream and
downstream from a splice donor site to a lentiviral gag initation
site. B. Envelope expression construct. An envelope construct
encoding vesicular stomatitis virus G glycoprotein (VSV-G) is
shown, though other non-lentiviral envelope proteins can be used
instead. Expression is driven by the HIV-1 LTR. The poly(A) site
was derived from the simian virus 40 late region. C. Transfer
vector constructs. In these constructs, Tat, Vpr, and Nef are
inactivated. Boxes interrupted by jagged lines contain partial
deletions. RRE=Rev-response element; .psi.=cis-acting packaging
signal; IRES=internal ribosome entry site; huCD25=human IL-2Ra
chain gene; GFP=Green Fluorescent Protein coding sequence;
RNAi=interfering RNA coding sequence; S=stop codon; T=termination
signal (e.g., SV40 polyA or BGH polyA); CS=cleavage site (e.g.,
viral 2A-like peptide cleaved by the 2A protease, presecretory
protein cleavage site, pancreatic prechymotrypsinogen cleavage
site); hPSA=human prostate specific antigen promoter; P1=promoter
1; CMV, Human CMV-IE promoter; P2=promoter-2; P53=tumor suppressor
gene; dsRNA=Bcl-2 RNAi (human).
[0037] FIG. 2 Heterologous Proteins. This table provides examples
of heterologous proteins contemplated for use in the vectors and
methods of the present invention.
[0038] FIG. 3 Transport Genes. This table provides examples of
transport genes contemplated for use in developing transport
sequences for the vectors and methods of the present invention.
[0039] FIG. 4 Promoter Elements. This table provides examples of
promoter elements contemplated for use in the vectors and methods
of the present invention.
[0040] FIG. 5 Enhancer Elements. This table provides examples of
enhancer elements contemplated for use in the vectors and methods
of the present invention.
[0041] FIG. 6 SIN-LV-P53-EGFP and SIN-LV-BCL2 RNAi-EGFP. The
drawing shows the vector constructs used as described in the
Examples.
[0042] FIG. 7 Therapeutic Constructs. This table provides examples
of constructs for use in the vectors and methods of the invention
for certain therapeutic applications.
[0043] FIG. 8 Infection of Prostate Cancer Cells. A. EGFP
expression in PC-3 cells infected with SIN-HIV-p53-IRES-EGFP (Panel
1) and PC-3 cells not infected with virus (2). B. EGFP expression
in 293T cells infected with SIN-HIV-p53-IRES-EGFP (Panel 1) and
293T cells not infected with virus (2). C. Viral packaging of
Vector. D. P53 expression. Lane 1: Size standards 1 kb plus DNA
Ladder. Lane 2: PC-3 cells (no infection). Lane 3: PC-3 cells
(infection). Lane 4: Blank. Lane 5: 293T cells (no infection). Lane
6: 293T cells (infection).
[0044] FIG. 9 Therapeutic Constructs 2. This table provides
examples of constructs for use in the vectors and methods of the
invention for certain therapeutic applications.
[0045] FIGS. 10A-10B Experimental evidence of efficacy of the viral
vector construct P53-Bcl-2 RNAi, expressing P53 and an RNAi agent
targeting human Bcl-2. FIGS. 10A and 10B show phenotype of in vitro
cell culture under phase-contrast microscope indicating that the
P53-Bcl-2 RNAi construct induces cell necrosis in PC3 prostate
cancer cells. A. untreated living cells. B. treated with viral
vector construct P53-Bcl-2 RNAi, expressing P53 and an RNAi agent
targeting human Bcl-2.
[0046] FIGS. 11A-11B Experimental evidence of efficacy of the viral
vector construct P53-Bcl-2 RNAi, expressing P53 and an RNAi agent
targeting human Bcl-2. FIGS. 11A and 11B show phenotype of in vitro
cell culture under phase-contrast microscope indicating that the
P53-Bcl-2 RNAi construct does not cause necrosis in 293T cells. A.
untreated cells. B. treated with viral vector construct P53-Bcl-2
RNAi, expressing P53 and an RNAi agent targeting human Bcl-2.
[0047] FIG. 12 In vivo test demonstrates tumor reduction efficacy
of viral vector construct P53-Bcl-2 RNAi, expressing P53 and an
RNAi agent targeting human Bcl-2. In order from left to right, far
left bar is weight of the control tumor group after three weeks;
tumor treated with P53 alone expressed through viral vector
construct (repair genetic defect); tumor treated with BCL2 SiRNA
alone expressed through viral vector construct (down regulation of
BCL2 gene) only; and on the far right tumor treated with viral
vector construct P53-Bcl-2 RNAi, expressing P53 and an RNAi agent
targeting human Bcl-2 for simultaneous P53 (repair) and BCL2
siRNA.
[0048] FIGS. 13A-13B FIGS. 13A and 13B show in vivo tumor staining
of control (untreated tumor) versus treatment groups.
Immunohistochemical staining by specific antibodies show that tumor
cells treated with viral vector construct P53-Bcl-2 RNAi,
expressing P53 and an RNAi agent targeting human Bcl-2 no longer
produce hPSA (human prostate specific antigen), which means that
the cells are no longer active tumor cells or are no longer
actively cancerous. A. control tumor expresses hPSA. B. treatment
tumor shows cell necrosis and no expreession of hPSA.
[0049] FIGS. 14A-14C FIGS. 14A-14C show a table that shows results
from another mouse (in vivo) study confirming tumor reduction
findings. Group V1 is prostrate tumor mice treated with P53
expressed alone in a viral vector; Group V2 is tumor mice treated
with BCL2 siRNA alone expressed through viral vector; V3 is tumor
mice treated with the dual viral construct expressing P53 and BCL2
siRNA. Group PC shows untreated tumor mice control. "**" in the
tables indicates tumor sizes on Day 7 as the starting sizes for
calculating the tumor growth rate and the ratio of Tumor
Weigh/Starting Tumor Size for groups of V1, V2, V3, and PC.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0050] In the present application, "a" and "an" are used to refer
to both single and a plurality of objects.
[0051] Unless otherwise indicated, all terms used herein have the
same ordinary meaning as they would to one skilled in the art of
the present invention.
[0052] Citation of documents herein is not intended as an admission
that any of the documents cited herein is pertinent prior art, or
an admission that the cited documents are considered material to
the patentability of the claims of the present application. All
statements as to the date or representations as to the contents of
these documents are based on the information available to the
applicant and do not constitute any admission as to the correctness
of the dates or contents of these documents.
[0053] As used herein, reference to "upstream", "downstream",
"first gene", "second gene", "last gene", "before", "after" and so
forth in relation to the spatial positioning of the various DNA
sequences in a vector, is meant to be with respect to the 5' to 3'
orientation of the vector sequence. For example "before" will have
the same meaning as "upstream of" or 5' of a particular reference
position, and "after" will have the same meaning of "downstream of"
or 3' with respect to a particular reference point on the
vector.
[0054] As used herein, "gene unit" includes a regulatory region
that may include a promoter and a heterologous nucleic acid
sequence encoding either an antisense RNA, RNAi or polypeptide of
interest that is controlled by the regulatory sequence, which is
typically referred to in the context of a multigene transfer
vector. However, in situations where a fused polypeptide is
desirous of being generated from the multigene vector of the
encoded polypeptides of adjacent gene units, the regulatory region
of the downstream gene units may include a nucleic acid sequence
encoding a cleavage site instead of a separate promoter.
MODES OF CARRYING OUT THE INVENTION
[0055] It is to be understood that this invention is not limited to
particular formulations or process parameters, as these may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments of
the invention only, and is not intended to be limiting. Further, it
is understood that a number of methods and materials similar or
equivalent to those described herein can be used in the practice of
the present invention.
[0056] I. Lentiviral Transfer System
[0057] The present invention provides a recombinant lentivirus
capable of infecting dividing and non-dividing cells. The virus is
useful for the in vivo and ex vivo transfer and expression of
nucleic acid sequences. Lentiviral vectors of the invention may be
lentiviral transfer plasmids or infectious lentiviral particles.
Construction of lentiviral vectors, helper constructs, envelope
constructs, etc., for use in lentiviral transfer systems has been
described, e.g., in U.S. Patent App. Pub. No. 2003/0119770,
"Intercellular delivery of a herpes simplex virus VP22 fusion
protein from cells infected with lentiviral vectors," incorporated
herein by reference in its entirety.
[0058] Lentiviruses
[0059] Lentiviruses are RNA viruses wherein the viral genome is
RNA. When a host cell is infected with a lentivirus, the genomic
RNA is reverse transcribed into a DNA intermediate which is
integrated very efficiently into the chromosomal DNA of infected
cells. This integrated DNA intermediate is referred to as a
provirus. Transcription of the provirus and assembly into
infectious virus occurs in the presence of an appropriate helper
virus or in a cell line containing appropriate sequences enabling
encapsidation without coincident production of a contaminating
helper virus. As described below, a helper virus is not required
for the production of the recombinant lentivirus of the present
invention, since the sequences for encapsidation are provided by
co-transfection with appropriate vectors.
[0060] The lentiviral genome and the proviral DNA have three genes:
the gag, the pol, and the env, which are flanked by two long
terminal repeat (LTR) sequences. The gag gene encodes the internal
structural (matrix, capsid, and nucleocapsid) proteins; the pol
gene encodes the RNA-directed DNA polymerase (reverse
transcriptase) and the env gene encodes viral envelope
glycoproteins. The 5' and 3' LTRs serve to promote transcription
and polyadenylation of the virion RNAs. The LTR contains all other
cis-acting sequences necessary for viral replication. Lentiviruses
have additional genes including vit, vpr, tat, rev, vpu, nef, and
vpx (in HIV-1, HIV-2 and/or SIV).
[0061] Adjacent to the 5' LTR are sequences necessary for reverse
transcription of the genome (the tRNA primer binding site) and for
efficient encapsidation of viral RNA into particles (the Psi site,
.psi.). If the sequences necessary for encapsidation (or packaging
of lentiviral RNA into infectious virions) are missing from the
viral genome, the result is a cis defect which prevents
encapsidation of genomic RNA. The resulting mutant is still capable
of directing the synthesis of all virion proteins, but lacks
function of replication.
[0062] In a first embodiment, the invention provides a recombinant
lentivirus capable of infecting a dividing or non-dividing cell.
The recombinant lentivirus comprises a nucleic acid sequence
containing a lentiviral packaging signal flanked by lentiviral
cis-acting nucleic acid sequences necessary for reverse
transcription and integration, a heterologous nucleic acid sequence
operably linked to a regulatory nucleic acid sequence, and a
nucleic acid sequence encoding an intercellular trafficking signal,
where the nucleic acid sequence encoding the intercellular
trafficking signal is fused in-frame with the heterologous nucleic
acid sequence, where the lentivirus does not contain either a
complete gag, pol, tat, rev, or env gene.
[0063] The recombinant lentivirus of the invention is therefore
genetically modified in such a way that some of the structural,
infectious genes of the native virus have been removed, and some
removed sequences replaced with a nucleic acid sequence to be
delivered to a target non-dividing cell. After infection of a cell
by the virus, the virus releases its nucleic acid into the cell and
the lentivirus genetic material can integrate into the host cell
genome. The transferred lentivirus genetic material is then
transcribed and translated, e.g., as dictated by the regulatory
sequences, into proteins within the host cell.
[0064] Lentiviral Vector Systems
[0065] The invention provides a method of producing a recombinant
lentivirus capable of infecting a dividing or non-dividing cell
comprising transfecting a suitable host cell with the following: a
transfer vector providing a nucleic acid encoding a lentiviral gag
and a lentiviral pol, where the gag and pol nucleic acid sequences
are operably linked to a heterologous regulatory nucleic acid
sequence and where the transfer vector is defective for nucleic
acid sequence encoding functional env protein and devoid of
lentiviral sequences both upstream and downstream from a splice
donor site to a gag initiation site of a lentiviral genome; an
envelope construct providing a nucleic acid encoding a
non-lentiviral env protein; and a helper construct providing a
nucleic acid sequence containing a lentiviral packaging signal
flanked by lentiviral cis-acting nucleic acid sequences for reverse
transcription and integration, and providing a cloning site for
introduction of a heterologous nucleic acid sequence operably
linked to a regulatory nucleic acid sequence and optionally to a
nucleic acid sequence encoding an intercellular trafficking signal,
where the nucleic acid sequence encoding the intercellular
trafficking signal is fused in-frame with the heterologous nucleic
acid sequence, where the helper construct does not contain either a
complete gag, pol, or env gene, and recovering the recombinant
lentivirus. An illustration of the individual vectors used in the
method of the invention is shown in FIG. 1.
[0066] The method of the invention includes the combination of a
minimum of three vectors in order to produce a recombinant virion
or recombinant lentivirus. For example, a vector of the invention
can include (a) the p53 gene product, expressed and driven by a
regulatory nucleic acid sequence to treat tumor cells or migrating
cells having a p53 gene mutation; (b) a specific siRNA driven by
second regulatory nucleic acid sequence to down-regulate tumor
activity of the tumor cells in target tissue or organs; wherein the
double or multiple gene system is able to enhance delivery efficacy
and therapeutic response. It is understood that in the vectors and
methods of the present invention, the relative positions in the
transfer vector of the therapeutic molecules--be they proteins,
RNAi or other types of antisense agents--can vary as needed.
Therefore, for example, any of the first, second, or third
heterologous nucleic acid sequences can encode an RNAi.
Furthermore, multiple heterologous nucleic acid sequences (e.g.,
two or three) can encode an RNAi or other antisense agent.
[0067] A first vector is a helper construct, which provides a
nucleic acid encoding a lentiviral gag and a lentiviral pol (FIG.
1A).
[0068] A second vector is an envelope construct, which provides a
nucleic acid encoding a non-lentiviral env protein (FIG. 1B). The
env gene can be derived from any virus excluding lentiviruses. The
env gene is ideally derived from a virus other than HIV. The env
gene may be amphotropic envelope protein which allows transduction
of cells of human and other species, or may be ecotropic envelope
protein, which is able to transduce only mouse and rat cells.
Further, it may be desirable to target the recombinant virus by
linkage of the envelope protein with an antibody or a particular
ligand for targeting to a receptor of a particular cell-type. By
inserting a sequence (including regulatory region) of interest into
the viral vector, along with another gene which encodes the ligand
for a receptor on a specific target cell, for example, the vector
is now target specific. Lentiviral vectors can be made target
specific by inserting, for example, a protein. Targeting is often
accomplished by using an antibody to target the lentiviral vector.
Those of skill in the art will know of, or can readily ascertain
without undue experimentation, specific methods to achieve delivery
of a lentiviral vector to a specific target.
[0069] Examples of retroviral-derived env genes include, but are
not limited to: Moloney murine leukemia virus (MoMuLV), Harvey
murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV),
gibbon ape leukemia virus (GaLV), and Rous Sarcoma Virus (RSV).
Other env genes such as Vesicular stomatitis virus (VSV) (Protein
G) can also be used.
[0070] The construct providing the viral env nucleic acid sequence
is operably associated with regulatory sequence, e.g., a promoter
or enhancer. Preferably, the regulatory sequence is a viral
promoter. The regulatory sequence can be any eukaryotic promoter or
enhancer, including for example, the Moloney murine leukemia virus
promoter-enhancer element, the human cytomegalovirus enhancer, or
the vaccinia P7.5 promoter. In some cases, such as the HIV-1
promoter-enhancer element, these promoter-enhancer elements are
located within or adjacent to the LTR sequences.
[0071] A third vector, the transfer vector, provides a nucleic acid
sequence, which contains the cis-acting viral sequences necessary
for the lentiviral life cycle. Such sequences include the
lentiviral psi packaging sequence, reverse transcription signals,
integration signals, viral promoter, enhancer, and polyadenylation
sequences. The transfer vector also contains a cloning site for a
heterologous nucleic acid sequence to be transferred to a dividing
or non-dividing cell, and optionally a nucleic acid sequence
encoding an intercellular trafficking signal, where the nucleic
acid sequence encoding the intercellular trafficking signal is
fused in-frame with the heterologous nucleic acid sequence (FIG.
1C).
[0072] Since recombinant lentiviruses produced by standard methods
in the art are defective, they require assistance in order to
produce infectious vector particles. Typically, this assistance is
provided, for example, by using a helper cell line that provides
the missing viral functions. These plasmids are missing a
nucleotide sequence which enables the packaging mechanism to
recognize an RNA transcript for encapsidation. Suitable cell lines
produce empty virions, since no genome is packaged. If a lentiviral
vector is introduced into such cells in which the packaging signal
is intact, but the structural genes are replaced by other genes of
interest, the vector can be packaged and vector virion
produced.
[0073] The method of producing the recombinant lentivirus of the
invention is different than the standard helper virus/packaging
cell line method described above. The three or more individual
vectors used to co-transfect a suitable packaging cell line
collectively contain all of the required genes for production of a
recombinant virus for infection and transfer of nucleic acid to a
non-dividing cell. Consequently, there is no need for a helper
virus.
[0074] Conveniently during the cloning stage, the nucleic acid
construct referred to as the transfer vector, having the packaging
signal and the heterologous cloning site, also contains a
selectable marker gene. Marker genes are utilized to assay for the
presence of the vector, and thus, to confirm infection and
integration. Typical selection genes encode proteins that confer
resistance to antibiotics and other toxic substances, e.g.
histidinol, puromycin, hygromycin, neomycin, methotrexate, etc.
[0075] "Non-dividing" cell refers to a cell that does not go
through mitosis. Non-dividing cells may be blocked at any point in
the cell cycle, (e.g., G.sub.0/G.sub.1, G.sub.1/S, G.sub.2/M), as
long as the cell is not actively dividing. For ex vivo infection, a
dividing cell can be treated to block cell division by standard
techniques used by those of skill in the art, including,
irradiation, aphidocolin treatment, serum starvation, and contact
inhibition. However, it should be understood that ex vivo infection
is often performed without blocking the cells since many cells are
already arrested (e.g., stem cells). The recombinant lentivirus
vector of the invention is capable of infecting any non-dividing
cell, regardless of the mechanism used to block cell division or
the point in the cell cycle at which the cell is blocked. Examples
of pre-existing non-dividing cells in the body include neuronal,
muscle, liver, skin, heart, lung, and bone marrow cells, and their
derivatives.
[0076] The method of the invention provides at least three vectors
which provide all of the functions required for packaging of
recombinant virions as discussed above. The method also envisions
transfection of vectors including viral genes such as vpr, vif,
nef, vpx, tat, rev, and vpu. Some or all of these genes can be
included, for example, on the packaging construct vector, or,
alternatively, they may reside on individual vectors. There is no
limitation to the number of vectors which are utilized, as long as
they are co-transfected to the packaging cell line in order to
produce a single recombinant lentivirus. For example, one could put
the env nucleic acid sequence on the same construct as the gag and
pol.
[0077] The vectors are introduced via transfection or infection
into the packaging cell line. The packaging cell line produces
viral particles that contain the vector genome. Methods for
transfection or infection are well known by those of skill in the
art. After co-transfection of the at least three vectors to the
packaging cell line, the recombinant virus is recovered from the
culture media and titered by standard methods used by those of
skill in the art.
[0078] In another embodiment, the invention provides a recombinant
lentivirus produced by the method of the invention as described
above.
[0079] The invention also provides a method of nucleic acid
transfer to a non-dividing cell to provide expression of a
particular nucleic acid sequence. Therefore, in another embodiment,
the invention provides a method for introduction and expression of
a heterologous nucleic acid sequence in a non-dividing cell
comprising infecting the non-dividing cell with the recombinant
virus of the invention and expressing the heterologous nucleic acid
sequence in the non-dividing cell.
[0080] It may be desirable to modulate the expression of a gene
regulating molecule in a cell by the introduction of a molecule by
the method of the invention. The term "modulate" envisions the
suppression of expression of a gene when it is over-expressed, or
augmentation of expression when it is under-expressed. Where a cell
proliferative disorder is associated with the expression of a gene,
nucleic acid sequences that interfere with the gene's expression at
the translational level can be used. This approach utilizes, for
example, antisense nucleic acid, ribozymes, or triplex agents,
siRNA to block transcription or translation of a specific mRNA,
either by masking that mRNA with an antisense nucleic acid or
triplex agent, or by cleaving it with a ribozyme.
[0081] The method of the invention may also be useful for neuronal
or glial cell transplantation, or "grafting," which involves
transplantation of cells infected with the recombinant lentivirus
of the invention ex vivo, or infection in vivo into the central
nervous system or into the ventricular cavities or subdurally onto
the surface of a host brain. Such methods for grafting will be
known to those skilled in the art and are described in Neural
Grafting in the Mammalian CNS, Bjorklund and Stenevi, eds. (1985).
Procedures include intraparenchymal transplantation, (i.e., within
the host brain) achieved by injection or deposition of tissue
within the host brain so as to be apposed to the brain parenchyma
at the time of transplantation.
[0082] Self-Inactivating Lentiviral Vectors
[0083] Self-inactivating (SIN) lentiviral vectors have a deletion
in the U3 region of the 3' LTR that eliminates regulatory
sequences, including the TATA box. The deletion has been reported
to result in transcriptional inactivation of the LTR in proviruses
without affecting vector titers or transgene expression in vitro.
SIN vectors are described, e.g., by Zufferey, et al., 1998, J.
Virology 72(12):9873-9880, who made a 400 by deletion, and Miyoshi,
et al., 1998, J. Virology 72(10):8150-8157, who made a 133 by
deletion.
[0084] It has been reported that a certain U3 deletion actually
results in increased expression from the vector in vivo (Bayer, et
al., 2008, A Large U3 Deletion Causes Increased In Vivo Expression
from a Nonintegrating Lentiviral Vector, Molecular Therapy
doi:10.1038/mt.2008.199). This finding suggests that additional
alterations to the lentivirus sequences are needed to ensure safety
of gene transfer systems.
[0085] II. Heterologous Nucleic Acid Sequences
[0086] A heterologous nucleic acid sequence is operably linked to a
regulatory nucleic acid sequence. As used herein, the term
"heterologous" nucleic acid sequence refers to a sequence that
originates from a foreign species, or, if from the same species, it
may be substantially modified from its original form.
Alternatively, an unchanged nucleic acid sequence that is not
normally expressed in a cell is a heterologous nucleic acid
sequence. The term "operably linked" refers to functional linkage
between the regulatory sequence and the heterologous nucleic acid
sequence. The heterologous sequence can be linked to a promoter.
The heterologous nucleic acid sequence can be under control of
either the viral LTR promoter-enhancer signals or of an internal
promoter, and retained signals within the lentiviral LTR can still
bring about efficient integration of the vector into the host cell
genome. The use of nonintegrating vectors for certain purposes,
e.g., where transient expression is sufficient, is also
contemplated.
[0087] The recombinant virus of the invention is capable of
transferring nucleic acid sequences into a non-dividing cell. The
term "nucleic acid sequence" refers to any nucleic acid molecule,
preferably DNA. The nucleic acid molecule may be derived from a
variety of sources, including DNA, cDNA, synthetic DNA, RNA, or
combinations thereof. Such nucleic acid sequences may comprise
genomic DNA which may or may not include naturally occurring
introns. Moreover, such genomic DNA may be obtained in association
with promoter regions, introns, or poly(A) sequences. Genomic DNA
may be extracted and purified from suitable cells by means well
known in the art. Alternatively, messenger RNA (mRNA) can be
isolated from cells and used to produce cDNA by reverse
transcription or other means.
[0088] FIG. 2 shows examples of heterologous proteins that can be
expressed from their genes using the vectors and methods of the
present invention. FIGS. 7 and 8 further list examples of cloned
structural genes that can serve as, e.g., a first, second, or third
heterologous nucleic acid sequence of the invention.
[0089] A preferred protein for expression using the vectors and
methods of the present invention is tumor antigen P53. Expression
of P53 is defective in most cancers, e.g., due to mutation of the
gene or lowered expression. Delivery of the wild-type gene encoding
the 53-kilodalton protein is therefore a goal of gene therapy for
many cancers.
[0090] Nucleic acids encoding the same proteins or targeting the
same RNAs can be used in a single transfer vector, for example, two
genes for the same protein can be cloned from different sources and
used as the first and second heterologous nucleic acid sequences.
Similarly, RNAi sequences that are specific for different parts of
the same target RNA, or that differ in their percent homology to
the target RNA, can be used together.
[0091] It may be desirable to transfer a nucleic acid encoding a
biological response modifier. Included in this category are
immunopotentiating agents including nucleic acids encoding a number
of the cytokines classified as "interleukins." These include, for
example, interleukins 1 through 12. Also included in this category,
although not necessarily working according to the same mechanisms,
are interferons, and in particular gamma interferon (.gamma.-IFN),
tumor necrosis factor (TNF) and granulocyte-macrophage-colony
stimulating factor (GM-CSF). It may be desirable to deliver such
nucleic acids to bone marrow cells or macrophages to treat
enzymatic deficiencies or immune defects, or cancer disease.
Nucleic acids encoding growth factors, toxic peptides, ligands,
receptors, or other physiologically important proteins can also be
introduced into specific non-dividing cells.
[0092] Selection of RNAi Agents and Other Antisense Nucleic Acid
Sequences
[0093] An RNAi agent used in the vectors and methods of the present
invention can be targeted to any RNA molecule. Besides messenger
RNA (mRNA), RNAi agents can target, e.g., various species of
microRNA. The use of RNAi in gene therapy and RNAi selection and
sequence design, are described, e.g., in WO 2007/109131,
"Lentiviral Vectors That Provide Improved Expression and Reduced
Variegation after Transgenesis," and WO 2007/087113, "Natural
Antisense and Non-Coding RNA Transcripts as Drug Targets," both of
which are incorporated herein by reference.
[0094] It may be desirable to modulate the expression of a gene
regulating molecule in a cell by the introduction of a molecule by
the method of the invention. The term "modulate" envisions the
suppression of expression of a gene when it is over-expressed, or
augmentation of expression when it is under-expressed. Where a cell
proliferative disorder is associated with the expression of a gene,
nucleic acid sequences that interfere with the gene's expression at
the translational level can be used. This approach utilizes, for
example, antisense nucleic acid, ribozymes, or triplex agents,
siRNA to block transcription or translation of a specific mRNA,
either by masking that mRNA with an antisense nucleic acid or
triplex agent, or by cleaving it with a ribozyme.
[0095] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, 1990 Scientific American 262:40). In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will not
translate an mRNA that is double-stranded. Antisense oligomers of
about 15 nucleotides are preferred, since they are easily
synthesized and are less likely to cause problems than larger
molecules when introduced into the target cell. The use of
antisense methods to inhibit the in vitro translation of genes is
well known in the art (Marcus-Sakura, 1988 Anal Biochem
172:289).
[0096] The antisense nucleic acid can be used to block expression
of a mutant protein or a dominantly active gene product, such as
amyloid precursor protein that accumulates in Alzheimer's disease.
Such methods are also useful for the treatment of Huntington's
disease, hereditary Parkinsonism, and other diseases. Antisense
nucleic acids are also useful for the inhibition of expression of
proteins associated with toxicity.
[0097] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al. 1991 Antisense Res and Dev 1:227; Helene, C.
1991 Anticancer Drug Design 6:569).
[0098] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, 1988 J Amer Med Assn
260:3030). A major advantage of this approach is that, because they
are sequence-specific, only mRNAs with particular sequences are
inactivated.
[0099] RNA interference (RNAi) is mediated by double stranded RNA
(dsRNA) molecules that have sequence-specific homology to their
target nucleic acid sequences (Caplen, N. J., et al, Proc. Natl.
Acad. ScL USA 98:9742-9747 (2001)). Biochemical studies in
Drosophila cell-free lysates indicate that, in certain embodiments
of the present invention, the mediators of RNA-dependent gene
silencing are 21-25 nucleotide "small interfering" RNA duplexes
(siRNAs). The siRNAs are derived from the processing of dsRNA by an
RNase enzyme known as Dicer (Bernstein, E., et al, Nature
409:363-366 (2001)). siRNA duplex products are recruited into a
multi-protein siRNA complex termed RISC (RNA Induced Silencing
Complex). Without wishing to be bound by any particular theory, a
RISC is then believed to be guided to a target nucleic acid
(suitably mRNA), where the siRNA duplex interacts in a
sequence-specific way to mediate cleavage in a catalytic fashion
(Bernstein, E., et al, Nature 409:363-366 (2001); Boutla, A., et
al, Curr. Biol. 11:1776-1780 (2001)). Small interfering RNAs that
can be used in accordance with the present invention can be
synthesized and used according to procedures that are well known in
the art and that will be familiar to the ordinarily skilled
artisan. Small interfering RNAs for use in the methods of the
present invention suitably comprise between about 0 to about 50
nucleotides (nt). In examples of nonlimiting embodiments, siRNAs
can comprise about 5 to about 40 nt, about 5 to about 30 nt, about
10 to about 30 nt, about 15 to about 25 nt, or about 20-25
nucleotides.
[0100] "RNAi" or "RNAi agent" refers to an at least partly
double-stranded RNA having a structure characteristic of molecules
that are known in the art to mediate inhibition of gene expression
through an RNAi mechanism or an RNA strand comprising at least
partially complementary portions that hybridize to one another to
form such a structure. When an RNA comprises complementary regions
that hybridize with each other, the RNA will be said to
self-hybridize. An RNAi agent includes a portion that is
substantially complementary to a target gene. An RNAi agent,
optionally includes one or more nucleotide analogs or
modifications. One of ordinary skill in the art will recognize that
RNAi agents that are synthesized in vitro can include
ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified
nucleotides or backbones, etc., whereas RNAi agents synthesized
intracellularly, e.g., encoded by DNA templates, typically consist
of RNA, which may be modified following transcription. Of
particular interest herein are short RNAi agents, i.e., RNAi agents
consisting of one or more strands that hybridize or self-hybridize
to form a structure that comprises a duplex portion between about
15-29 nucleotides in length, optionally having one or more
mismatched or unpaired nucleotides within the duplex. RNAi agents
include short interfering RNAs (siRNAs), short hairpin RNAs
(shRNAs), and other RNA species that can be processed
intracellularly to produce shRNAs including, but not limited to,
RNA species identical to a naturally occurring miRNA precursor or a
designed precursor of an miRNA-like RNA.
[0101] The term "short, interfering RNA" (siRNA) refers to a
nucleic acid that includes a double-stranded portion between about
15-29 nucleotides in length and optionally further comprises a
single-stranded overhang {e.g., 1-6 nucleotides in length) on
either or both strands. The double-stranded portion is typically
between 17-21 nucleotides in length, e.g., 19 nucleotides in
length. The overhangs are typically present on the 3' end of each
strand, are usually 2 nucleotides long, and are composed of DNA or
nucleotide analogs. An siRNA may be formed from two RNA strands
that hybridize together, or may alternatively be generated from a
longer double-stranded RNA or from a single RNA strand that
includes a self-hybridizing portion, such as a short hairpin RNA.
One of ordinary skill in the art will appreciate that one or more,
mismatches or unpaired nucleotides can be present in the duplex
formed by the two siRNA strands. One strand of an siRNA (the
"antisense" or "guide" strand) includes a portion that hybridizes
with a target nucleic acid, e.g., an mRNA transcript. Typically the
antisense strand is perfectly complementary to the target over
about 15-29 nucleotides, typically between 17-21 nucleotides, e.g.,
19 nucleotides, meaning that the siRNA hybridizes to the target
transcript without a single mismatch over this length. However, one
of ordinary skill in the art will appreciate that one or more
mismatches or unpaired nucleotides may be present in a duplex
formed between the siRNA strand and the target transcript.
[0102] "Short hairpin RNA" refers to a nucleic acid molecule
comprising at least two complementary portions hybridized or
capable of hybridizing to form a duplex structure sufficiently long
to mediate RNAi (typically between 15-29 nucleotides in length),
and at least one single-stranded portion, typically between
approximately 1 and 10 nucleotides in length that forms a loop
connecting the ends of the two sequences that form the duplex. The
structure may further comprise an overhang. The duplex formed by
hybridization of self-complementary portions of the shRNA has
similar properties to those of siRNAs and, as described below,
shRNAs are processed into siRNAs by the conserved cellular RNAi
machinery. Thus shRNAs are precursors of siRNAs and are similarly
capable of inhibiting expression of a target transcript. As is the
case for siRNA, an shRNA includes a portion that hybridizes with a
target nucleic acid, e.g., an mRNA transcript and is usually the
perfectly complementary to the target over about 15-29 nucleotides,
typically between 17-21 nucleotides, e.g., 19 nucleotides. However,
one of ordinary skill in the art will appreciate that one or more
mismatches or unpaired nucleotides may be present in a duplex
formed between the shRNA strand and the target transcript.
[0103] An RNAi agent is considered to be "targeted" to a transcript
and to the gene that encodes the transcript if (1) the RNAi agent
comprises a portion, e.g., a strand, that is at least approximately
80%, approximately 85%, approximately 90%, approximately 91%,
approximately 92%, approximately 93%, approximately 94%,
approximately 95%, approximately 96%, approximately 97%,
approximately 98%, approximately 99%, or approximately 100%
complementary to the transcript over a region about 15-29
nucleotides in length, e.g., a region at least approximately 15,
approximately 17, approximately 18, or approximately 19 nucleotides
in length; and/or (2) the Tm of a duplex formed by a stretch of 15
nucleotides of one strand of the RNAi agent and a 15 nucleotide
portion of the transcript, under conditions (excluding temperature)
typically found within the cytoplasm or nucleus of mammalian cells
and/or in a Drosophila lysate as described, e.g., in U.S. Patent
App. Pubs. 2002/0086356 and 2004/0229266, is no more than
approximately 15.degree. C. lower or no more than approximately
10.degree. C. lower, than the Tm of a duplex that would be formed
by the same 15 nucleotides of the RNAi agent and its exact
complement; and/or (3) the stability of the transcript is reduced
in the presence of the RNAi agent as compared with its absence. An
RNAi agent targeted to a transcript is also considered targeted to
the gene that encodes and directs synthesis of the transcript. A
"target region" is a region of a target transcript that hybridizes
with an antisense strand of an RNAi agent. A "target transcript" is
any RNA that is a target for inhibition by RNA interference. The
terms "target RNA" and "target transcript" are used interchangeably
herein.
[0104] Selection of appropriate RNAi agents is facilitated by using
computer programs that automatically align nucleic acid sequences
and indicate regions of identity or homology. Such programs are
used to compare nucleic acid sequences obtained, for example, by
searching databases such as GenBank or by sequencing PCR products.
Comparison of nucleic acid sequences from a range of species allows
the selection of nucleic acid sequences that display an appropriate
degree of identity between species. In the case of genes that have
not been sequenced, Southern blots are performed to allow a
determination of the degree of identity between genes in target
species and other species. By performing Southern blots at varying
degrees of stringency, as is well known in the art, it is possible
to obtain an approximate measure of identity. These procedures
allow the selection of RNAi that exhibit a high degree of
complementarity to target nucleic acid sequences in a subject to be
controlled and a lower degree of complementarity to corresponding
nucleic acid sequences in other species. One skilled in the art
will realize that there is considerable latitude in selecting
appropriate regions of genes for use in the present invention.
[0105] Selection of an appropriate antisense nucleic acid is
facilitated by using computer programs that automatically align
nucleic acid sequences and indicate regions of identity or
homology. Such programs are used to compare nucleic acid sequences
obtained, for example, by searching databases such as GenBank or by
sequencing PCR products. Comparison of nucleic acid sequences from
a range of species allows the selection of nucleic acid sequences
that display an appropriate degree of identity between species. In
the case of genes that have not been sequenced, Southern blots can
be performed to allow a determination of the degree of identity
between genes in target species and other species. By performing
Southern blots at varying degrees of stringency, as is well known
in the art, it is possible to obtain an approximate measure of
identity. These procedures allow the selection of antisense nucleic
acids that exhibit a high degree of complementarity to target
nucleic acid sequences in a subject to be controlled and a lower
degree of complementarity to corresponding nucleic acid sequences
in other species. One skilled in the art will realize that there is
considerable latitude in selecting appropriate regions of genes for
use in the present invention.
[0106] Selection of hybridization sites for antisense nucleic acids
can be made by one of skill in the art using methods described in
the literature. For example, Ding, et al., report a method for
defining mRNA hybridization sites based on determining RNA
structures using algorithms and thermodynamic and structural
properties of the RNA (Ding, et al., 2001, Statistical prediction
of single-stranded regions in RNA secondary structure and
application to predicting effective antisense target sites and
beyond, Nucleic Acids Research 29(5):1034-1046; incorporated herein
by reference in its entirety). Sczakiel, et al., also describe a
method for computer-supported design of antisense oligonucleotides
(Sczakiel, et al., 2000, Theoretical and experimental approaches to
design effective antisense oligonucleotides, Frontiers in
Bioscience 5: D194-201; Schen, et al., 2000, RNA accessibility
prediction: a theoretical approach is consistent with experimental
studies in cell extracts, Nucleic Acids Research 28: 2455-2461;
Patzel, et al., 1999, A theoretical approach to select effective
antisense oligodeoxyribonucleotides at high statistical
probability, J. Biol. Chem. 266:18162-18171; all incorporated
herein by reference in their entirety).
[0107] Reports of other methods for identifying mRNA hybridization
sites used include, e.g., Chiang, et al., who describe a method
based on calculating melting temperatures (Chiang, et al., 1991,
Antisense oligonucleotides inhibit intercellular adhesion molecule
1 expression by two distinct mechanisms, J. Biol. Chem. 266:
18162-18171, incorporated herein by reference in its entirety).
Methods based on calculation of duplex formation free energies have
been used (see, e.g., Stull, et al., 1992, Predicting antisense
oligonucleotide inhibitory efficacy: a computational approach using
histograms and thermodynamic indices, Nucleic Acids Research
20:3501-3508; Ding, et al., 1999, A bayesian statistical algorithm
for RNA secondary structure prediction, Comput. Chem. 23:387-400;
all incorporated herein by reference in their entirety). Still
other methods rely on the use of combinatorial oligonucleotides to
identify the hybridization sites within the target RNA.
Identification of the hybridization sites is made using RNase H
cleavage (Lloyd, et al., 2001, Determination of optimal sites of
antisense oligonucleotide cleavage within TNF.alpha. mRNA, Nucleic
Acids Research 29:3664-3673, incorporated herein by reference in
its entirety), microarray analysis (Mir, et al., 1999, Determining
the influence of structure on hybridization using oligonucleotide
arrays, Nature Biotechnology 17:788-792; and Sohail, et al., 2001,
Antisense oligonucleotides selected by hybridization to scanning
arrays are effective reagents in vivo, Nucleic Acids Research
29:2041-2051, both incorporated herein by reference in their
entirety) or MALDI-TOF mass spectrometry (Altman, et al., 1999,
Selection of modified oligonucleotides with increased target
affinity via MALDI-monitored nuclease survival assays, J. Comb.
Chem. 1:493-508, incorporated herein by reference in its
entirety).
[0108] The utility of an antisense nucleic acid molecule for
modulation (including inhibition) of an mRNA can be readily
determined by simple testing. Thus, an in vitro or in vivo
expression system comprising the targeted mRNA, mutations or
fragments thereof, can be contacted with a particular antisense
nucleic acid molecule (modified or un modified) and levels of
expression are compared to a control, that is, using the identical
expression system which was not contacted with the antisense
nucleic acid molecule. In vitro assays of oligonucleotide activity
can also be useful for identifying antisense nucleic acids of the
invention. For example, Lloyd, et al., report a direct inverse
correlation between predicted chimeric antisense oligonucleotide
activities, as determined using an in vitro RNase H assay, and the
resultant levels of mRNA and protein expression (Lloyd, et al.,
2001, Determination of optimal sites of antisense oligonucleotide
cleavage within TNF.alpha. mRNA, Nucleic Acids Research 29(17):
3664-3673). According to Lloyd, et al., the ability of the in vitro
assay to predict oligonucleotide efficacy was superior to other
computationally based RNA structural predictions, .DELTA.G
calculations and in vivo trial and error methodologies.
[0109] Bcl-2 and molecules that work in conjunction with Bcl-2 are
also targets for cancer therapy. B cell leukemia/lymphoma-2 (Bcl-2)
is the prototype member of a family of cell death regulatory
proteins. Bcl-2 is found mainly in the mitochondria and blocks
apoptosis by interfering with the activation of caspases. Gene
transfer of Bcl-2 into tumor cells has been shown to enhance their
metastatic potential (Miyake et al., 1999). Bcl-2 gene transfer may
be applied to bone marrow transplant since Bcl-2 enhances the
survival of hematopoietic stem cells after reconstitution of
irradiated recipient (Innes et al., 1999). Also, Bcl-2 gene
transfer could be useful against neurodegenerating diseases since
expression of Bcl-2 in neurons protects them from apoptosis (Saille
et al., 1999). Bcl-XS (short isoform) is a dominant negative
repressor of Bcl-2 and Bcl-XL. It has been used in gene therapy
experiments to initiate apoptosis in tumors that express Bcl-2 and
Bcl-XL. Expression of Bcl-XS reduces tumor size (Ealovega et al.,
1996) and sensitizes tumor cells to chemotherapeutic agents
(Sumatran et al., 1995), suggesting a role for Bcl-XS in initiating
cell death in tumors that express Bcl-2 or Bcl-XL (Dole et al.,
1996). Expression of these genes or RNAi agents targeting them can
be selected as appropriate for the condition being treated.
[0110] Equivalent Molecules
[0111] The invention comprehends that the therapeutic molecules
delivered using the vectors and methods of the present invention
can be modified. The nucleic acid molecule encoding a given protein
be modified, for instance, due to the degeneracy of codon usage, a
coding sequence can be modified, and modified and truncated forms
of a protein can be used, such as those which may be found in the
literature or analogous to truncated or modified forms found in the
literature.
[0112] Likewise, analogs, homologs, derivatives, and variants of
the coding sequences can be used and analogs, homologs, derivatives
and variants of proteins can be expressed; such expressed analogs,
homologs, derivatives and variants of proteins can have activity
analogous to that of the full-length protein, and the analogs,
homologs, derivatives and variants of the protein coding sequence
encode such active analogs, homologs, derivatives, and
variants.
[0113] III. Heterologous Regulatory Sequences
[0114] A "first heterologous regulatory sequence" is positioned
upstream (5' of) the first heterologous nucleic acid sequence,
encoding, e.g., a therapeutic gene. Similarly, a "second
heterologous regulatory sequence" can be positioned upstream (5'
of) the second heterologous nucleic acid sequence and downstream of
the first heterologous nucleic acid sequence, and a "third
heterologous regulatory sequence" can be positioned upstream (5'
of) the third heterologous nucleic acid sequence and downstream of
the second heterologous nucleic acid sequence. A heterologous
regulatory sequence can be a sequence that influences, e.g.,
expression or localization, of a therapeutic molecule encoded by a
heterologous nucleic acid sequence. A heterologous regulatory
sequence can comprise a promoter, enhancer, protease recognition
(cleavage) sequence, internal ribosome binding site, intracellular
or intercellular trafficking (transport) signal, etc. Certain
heterologous regulatory sequences, e.g., cleavage sequences and
trafficking sequences, can be expressed as part of a fusion with a
therapeutic molecule.
[0115] In embodiments, a heterologous regulatory sequence affects
an upstream heterologous nucleic acid sequence. Therefore, a fourth
regulatory sequence, located downstream of a third heterologous
nucleic acid sequence, can be included in the transfer vector.
Also, for example, the second heterologous regulatory sequence can
contain elements that affect expression or localization of the
first heterologous nucleic acid and its corresponding therapeutic
molecule, and the third heterologous regulatory sequence can
contain elements that affect expression or localization of the
second heterologous nucleic acid and its corresponding therapeutic
molecule.
[0116] Promoters and Enhancers
[0117] The promoter sequence may be homologous or heterologous to
the desired gene sequence. A wide range of promoters may be
utilized, including viral or mammalian promoters. Cell or tissue
specific promoters can be utilized to target expression of gene
sequences in specific cell populations. Suitable mammalian and
viral promoters for the present invention are available in the
art.
[0118] Examples of promoters, cellular promoters/enhancers and
inducible promoters/enhancers that can be used in combination with
the present invention are listed in FIGS. 4 and 5. Any suitable
promoter/enhancer combination (as per the Eukaryotic Promoter
Database, EPDB) can be used to drive expression of constructs of
the invention.
[0119] Trafficking Signals
[0120] Trafficking signals can direct a molecule to different
compartments within a cell, as well as outside the cell and into
other cells. An "intercellular trafficking signal," or transport
signal, is an amino acid sequence that imparts the property to a
protein of being able to pass through membranes between cells.
Examples of membrane-penetrating proteins include, but are not
limited to, several plant and bacterial protein toxins, such as
ricin, abrin, modeccin, diphtheria toxin, cholera toxin, anthrax
toxin, heat labile toxins, and Pseudomonas aeruginosa exotoxin A.
Examples of membrane-penetrating proteins that are not toxins
include the TAT protein of human immunodeficiency virus and the
protein VP22, the product of the UL49 gene of herpes simplex virus
type 1. One line of research involves adapting such molecules from
their naturally destructive role into therapeutic compositions.
[0121] The effectiveness of lentiviral vectors to deliver genes
encoding proteins fused to herpes simplex virus type 1 tegument
protein VP22 has been reported (see, e.g., U.S. Pat. App. Pub. No.
2003/0119770). "VP22" denotes: protein VP22 of HSV, e.g., of HSV1,
and transport-active fragments and homologues thereof, including
transport-active homologues from other herpesviruses including
varicella zoster virus VZV, marek's disease virus MDV and bovine
herpesvirus BHV.
[0122] Among sub-sequences of herpesviral VP22 protein with
transport activity, investigators have found that, for example,
transport activity is present in polypeptides corresponding to
amino acids 60-301 and 159-301 of the full HSV1 VP22 sequence
(1-301). A polypeptide consisting of aa 175-301 of the VP22
sequence has markedly less transport activity, and is less
preferred in connection with the present invention. Accordingly,
the present invention relates in one aspect to a sub-sequence of
VP22 containing a sequence starting preferably from about aa 159
(or earlier, towards the N-terminal, in the native VP22 sequence),
to about aa 301, and having (relative to the full VP22 sequence) at
least one deletion of at least part of the VP22 sequence which can
extend for example from the N-terminal to the cited starting point,
e.g., a deletion of all or part of the sequence of about aa 1-158.
(Less preferably, such a deletion can extend further in the
C-terminal direction, e.g., to about aa 175.) For example, partial
sequences in the range from about aa 60-301 to about aa 159-301 are
provided.
[0123] VP22 sequences as contemplated herein extend to homologous
proteins and fragments based on sequences of VP22 protein
homologues from other herpesviruses, e.g., the invention provides
corresponding derivatives and uses of the known VP22-homologue
sequences from VZV (e.g., all or homologous parts of the sequence
from aa 1-302), from MDV (e.g., all or homologous parts of the
sequence from aa 1-249) and from BHV (e.g., all or homologous parts
of the sequence from aa 1-258). The sequences of the corresponding
proteins from HSV2, VZV, BHV and MDV are available in public
protein/nucleic acid sequence databases. Thus, for example, within
the EMBL/Genbank database, a VP22 sequence from HSV2 is available
as gene item UL49 under accession no. Z86099 containing the
complete genome of HSV2 strain HG52; the complete genome of VZV
including the homologous gene/protein is available under accession
numbers X04370, M14891, M16612; the corresponding protein sequence
from BHV is available as "bovine herpesvirus 1 virion tegument
protein" under accession number U21137; and the corresponding
sequence from MDV is available as gene item UL49 under accession
number L10283 for "gallid herpesvirus type 1 homologous sequence
genes". In these proteins, especially those from HSV2 and VZV,
corresponding deletions can be made, e.g., of sequences homologous
to aa 1-159 of VP22 from HSV1. Homologies between these sequences
are readily accessible by the use of standard algorithms, default
parameters, and software.
[0124] Furthermore, chimeric VP22 proteins and protein sequences
are also useful within the context of the present invention, e.g.,
a protein sequence from VP22 of HSV1 for part of which a homologous
sequence from the corresponding VP22 homologue of another
herpesvirus has been substituted. For example, into the sequence of
polypeptide 159-301 from VP22 of HSV1, C-terminal sequences can be
substituted from VP22 of HSV2 or from the VP22 homologue of
BHV.
[0125] Investigators have found that deletion of the 34-amino acid
C-terminal sequence from VP22 of HSV1 abolishes transport-activity,
thus this sequence region contains essential elements for transport
activity. According to a further aspect of the invention, there are
provided in-frame fusions comprising a nucleic acid sequence
encoding the 34-amino acid C-terminal sequence from VP22, or a
variant thereof, together with a sequence for a heterologous
nucleic acid sequence. In-frame fusions of nucleic acid sequences
encoding modified terminal fragments having at least one mutation
insertion or deletion relative to the C-terminal 34 amino acid
sequence of HSV1 VP22 are also provided.
[0126] Investigators have also been found that sequences necessary
for transport activity contain one or a plurality of amino acid
sequence motifs or their homologues from the C-terminal sequence of
VP22 of HSV1 or other herpesviruses, which can be selected from
RSASR (SEQ ID NO: 1), RTASR (SEQ ID NO: 2), RSRAR (SEQ ID NO: 3),
RTRAR (SEQ ID NO: 4), ATATR (SEQ ID NO 5), and wherein the third or
fourth residue A can be duplicated, e.g., as in RSAASR (SEQ ID NO:
6). Corresponding in-frame fusions of nucleic acid sequences
encoding these signals are also provided.
[0127] The HIV-1 Tat protein was also reported to enhance
intercellular trafficking in vitro. It is composed of 86 amino
acids and contains a highly basic region and a cysteine-rich
region. It was found that Tat-derived peptides as short as eleven
amino acids are sufficient for transduction of proteins (Fawell, et
al., 1994, Proc Natl Acad Sci USA 91:664-668). However, the exact
mechanism by which the 11-amino acid transduction domain crosses
lipid bilayers is poorly understood. Schwarze et al. (Science,
385:1569-1572, 1999) reported generating a Tat-.beta.-galactosidase
fusion protein that was delivered efficiently into brain tissue and
skeletal muscle in vivo.
[0128] In embodiments of the present invention, a Tat-derived
trafficking protein of eleven amino acids
Try-Gly-Arg-Lys-lys-Arg-Arg-Gln-Arg-Arg (SEQ ID NO: 7) is used to
enhance intercellular trafficking of therapeutic molecules. Any
appropriate nucleic acid sequence can be used to express this
protein, e.g., tat ggc agg aag aag cgg aga cag cga cga aga (SEQ ID
NO:8) with a start codon.
[0129] Control of intracellular as well as intercellular transport
is contemplated for use in the methods of the invention. This level
of control can be used to target therapeutic proteins to particular
cellular compartments, e.g., to correct defects for proteins
involved in specific disease processes.
[0130] Other potentially useful Tat-derived trafficking sequences
have been described. For example Chauhan, et al., 2007, The Taming
of the Cell Penetrating Domain of the HIV Tat: Myths and Realities,
J. Control Release 117(2): 148-162, incorporated herein by
reference in its entirety, disclose variants of the Tat protein
transduction domain.
[0131] Following are Tat-derived cell penetrating peptides
described by Chauhan, et al.:
TABLE-US-00001 PTD YGRKKRRQRRR (SEQ ID NO: 9) PTD-4 YARAAARQARA
(SEQ ID NO: 10) YM-3 THRLPRRRRRR (SEQ ID NO: 11) CTP GGRRARRRRRR
(SEQ ID NO: 12)
[0132] As reported by the authors, depending on the nature of the
protein being transported, these peptides effect transport,
including transport among cellular compartments. For example,
cytoplasmic proteins reportedly end up in the nucleus when PTD is
used, nucleo-cytoplasmic proteins go to the nucleus when PTD-4 is
used, secretory proteins are found in the nucleus and outside the
cell when YM-3 is used, and membrane proteins go to the membrane
and nucleus when CTP is used.
[0133] Other cell penetrating proteins useful for introducing
recombinant proteins into cells are penetratin, polylysine,
polyarginine, Kaposi FGF, Syn B1, FGF-4, nuclear localization
signal, anthrax toxin derivative 254-amino acids peptide segment,
diphtheria toxin "R" binding domain, MPG (described below), WR
peptide, and exotoxin A. Penetratin peptide has also been used for
siRNA delivery to cells. In embodiments, these proteins or their
derivatives used in as trafficking signals in the vectors and
methods of the invention.
[0134] A fusion peptide, "MPG," has been described for efficient
transduction of nucleic acids. This peptide is a bipartite
amphipathic peptide obtained by combining the fusion domain of
HIV-gp41 protein and the NLS domain of SV40 large T antigen. This
peptide is being used as a nanoparticle for transduction of siRNA
in vitro and is also available commercially. (See, e.g., Chauhan,
et al., 2007; Morris, et al., 1999, A novel potent strategy for
gene delivery using a single peptide vector as a carrier, Nucleic
Acids Research 27:3510-3517.)
[0135] In embodiments of the vectors and methods of the invention,
an siRNA or other antisense agent is transported intercellularly or
intracellularly.
[0136] The inventive transfer vector may include splice acceptor
and splice donor sequences flanking the gene construct. In the case
of multigene transfer vector, the splice acceptor sequence may be
inserted upstream of the first promoter-structural gene unit and
splice donor site may be located in the downstream of the last
promoter-structural gene unit in the 5' to 3' order. A mammalian
insulator sequence (MIS) may be inserted downstream of the gene
units, or if specific promoters are used for the gene units, then
the MIS may be placed before the first gene unit and after the last
gene unit followed by splice donor site. A translation initiation
sequence may also be included in the multigene gene transfer
vector. The use of the translation initiation sequence causes the
second and subsequent multigene units to be expressed evenly and
stably compared with the first gene expression product.
[0137] Translation Initiation Sequence
[0138] The transfer vector may optionally comprise a sequence that
allows for translation initiation in the middle of a messenger RNA
(mRNA) sequence as part of the greater process of protein
synthesis. While this sequence may be typically IRES other
sequences may be used, which may have a similar sequence.
[0139] Splice Acceptor/Splice Donor Sequence
[0140] The transfer vector may optionally comprise a promoter-gene
sequence flanked by a splice acceptor site 5' to a gene unit and a
splice donor site 3' to the promoter-gene. For instance, the
following elements may be present 5' to 3': a splice acceptor site,
first promoter for the first gene unit, first heterologous nucleic
acid sequence and splice donor site 3' to the first gene unit, then
a splice acceptor site and a second promoter for the second gene,
the second heterologous nucleic acid sequence, splice donor site,
and then if there is a third gene, a splice acceptor site, third
promoter for the third gene unit, the third heterologous nucleic
acid sequence, and a splice donor site, and so forth. The splice
acceptor or donor site typically may include about 5 to 10
bases.
[0141] Mammalian Insulator Sequence (MIS)
[0142] The transfer vector may optionally comprise an insulator
sequence, in particular mammalian insulator sequence (MIS).
Insulators are DNA sequence elements that can protect against the
activation influence of distal enhancers associated with other
genes, and also help to preserve the independent function of genes
embedded in a genome in which they are surrounded by regulatory
signals so that cross interaction is avoided. The insulators as
used in the present application may not necessarily be limited to
use in lentiviruses. The insulators may be used with other viral
vectors.
[0143] To provide background on these insulator sequences, the zinc
finger protein CCCTC-binding factor (CTCF) is a versatile
transcription regulator that binds to insulators and shows
enhancer-blocking activity for regulating gene expression control.
Chicken (beta-globin) insulator with about 1.2 kb is widely used in
vitro or in vivo animals, but generally does not have human or
mammalian compatible factors to be entirely useful in treating
humans or mammals.
[0144] Bovine or human growth hormone transcriptional stop
sequences may also be used as insulators. A short-element of about
238 by containing the "HS" DNA element core sequence, which is the
binding site for CTCF is also effective as an "enhancer blocking"
element. Template DNA for generation of such element, for example a
pcDNA3, which contains bovine growth hormone transcriptional stop
sequence may be as follows: PCR primers:
5'-agctagatagtgtcacctaaatgc-3' (SEQ ID NO:13) and
5'-agcatgcctgctatt-3' (SEQ ID NO:14).
[0145] A binding site for the transcription factor CTCF may be
responsible for enhancer-blocking activity in a variety of
insulators, including the insulators at the 5' and 3' chromatin
boundaries of the chicken and human beta-globin locus. The minimal
element responsible for this activity may be a binding site for
CTCF.
[0146] When several different sequences of insulators of 5' and 3'
HS/CTCF are compared using "human insulator" as the template, there
are at least two mutations in the mouse, at least 5 mutations in
chicken at the 5' HS/CTCF. In addition, there are at least two
mutations in mouse, at least 4 mutations at the 3' HS/CTCF.
[0147] The insulator sequence as used in the lentivirus transfer
vector may be placed as follows. In a single gene vector where a
general promoter is used to control gene expression, MIS may be
placed 3' of the gene and upstream of the splicing donor site.
However, if a specific promoter is used to control the expression
of the gene, the MIS may be placed upstream of the specific
promoter and downstream of the gene, wherein the MIS sequences are
optionally flanked by the splice acceptor on the 5' side and splice
donor on the 3' side of the gene. In a single gene construct, for
RNAi expression where specific promoter is used, MIS may flank the
specific promoter-structural gene construct, optionally with a
splice donor site downstream of the 3' MIS. Two or more MIS may be
used together to enhance the blocking effects.
[0148] For multiple gene vectors, if a general promoter is used, an
MIS may be included downstream of the gene units with splice donor
site 3' to the MIS. However, if a specific promoter is used MIS is
place upstream of the first specific promoter for the first
structural gene, and another MIS downstream of the last specific
promoter-structural gene set. The MIS are optionally flanked by
splice acceptor on the 5' side and splice donor on the 3' side.
[0149] IV. Therapeutic Applications
[0150] The invention includes a variety of therapeutic applications
for the lentiviral vectors of the invention. In particular,
lentiviral vectors are useful for gene therapy. Exemplary
therapeutic applications are listed in FIG. 7. The invention
provides methods of treating and/or preventing infection by an
infectious agent, the method comprising administering to a subject
prior to, simultaneously with, or after exposure of the subject to
the infectious agent a composition comprising an effective amount
of a lentiviral vector, wherein the lentiviral vector directs
transcription of at least one RNA that hybridizes to form an shRNA
or siRNA that is targeted to a transcript produced during infection
by the infectious agent, which transcript is characterized in that
reduction in levels of the transcript delays, prevents, and/or
inhibits one or more aspects of infection by and/or replication of
the infectious agent.
[0151] The invention provides methods of treating a disease or
clinical condition by, for example, removing a population of cells
from a subject at risk of or suffering from the disease or clinical
condition and engineering or manipulating the cells to comprise an
effective amount of therapeutic agents by infecting or transfecting
the cells with a lentiviral vector. At least a portion of the cells
are returned to the subject.
[0152] Without limitation, therapeutic approaches may find
particular use in diseases such as cancer, in which a mutation in a
cellular gene is responsible for or contributes to the pathogenesis
of the disease, and in which specific inhibition of the target
transcript bearing the mutation may be achieved by expressing an
RNAi agent targeted to the target transcript within the cells,
without interfering with expression of the normal (i.e.
non-mutated) allele. Furthermore, treatment of any cancer in which
P53 expression is defective is contemplated using the vectors and
methods of the invention.
[0153] The invention is also useful for the treatment of genetic
diseases, for example, Gaucher's Disease and Fabry's Disease.
Gaucher's disease is a lysosomal storage disease caused by a
deficiency of the enzyme glucocerebrosidase. This deficiency leads
to an accumulation of the enzyme substrate, the fatty substance
glucocerebroside (also known as glucosylceramide). Fatty material
can collect in the spleen, liver, kidneys, lungs, brain and bone
marrow. It has been reported, using a mouse model for Gaucher's
Disease, that a lentiviral vector can transduce HSCs that are
capable of long-term gene expression in vivo (Kim, et al., 2005,
"Long-term expression of the human glucocerebrosidase gene in vivo
after transplantation of bone-marrow-derived cells transformed with
a lentivirus vector, J. Gene Med. 7:878-887, incorporated herein by
reference).
[0154] According to certain embodiments of the invention, rather
than removing cells from the body of a subject, infecting or
transfecting them in tissue culture, and then returning them to the
subject, inventive lentiviral vectors or lentiviruses are delivered
directly to the subject.
[0155] Neurological Disorders
[0156] Cells infected with a recombinant lentivirus of the
invention, in vivo, or ex vivo, used for treatment of a neuronal
disorder for example, may optionally contain an exogenous gene, for
example, a gene which encodes a receptor or a gene which encodes a
ligand. Such receptors include receptors which respond to dopamine,
GABA, adrenaline, noradrenaline, serotonin, glutamate,
acetylcholine and other neuropeptides, as described above. Examples
of ligands which may provide a therapeutic effect in a neuronal
disorder include dopamine, adrenaline, noradrenaline,
acetylcholine, gamma-aminobutyric acid and serotonin. The diffusion
and uptake of a required ligand after secretion by an infected
donor cell would be beneficial in a disorder where the subject's
neural cell is defective in the production of such a gene product.
A cell genetically modified to secrete a neurotrophic factor, such
as nerve growth factor (NGF), might be used to prevent degeneration
of cholinergic neurons that might otherwise die without treatment.
Alternatively, cells can be grafted into a subject with a disorder
of the basal ganglia, such as Parkinson's disease, or can be
modified to contain an exogenous gene encoding L-DOPA, the
precursor to dopamine. Parkinson's disease is characterized by a
loss of dopamine neurons in the substantia nigra of the midbrain,
which have the basal ganglia as their major target organ.
[0157] U.S. Pat. No. 6,800,281, "Lentiviral-mediated growth factor
gene therapy for neurodegenerative diseases," incorporated herein
by reference in its entirety, describes methods for treating
Parkinson's Disease using glial cell derived neurotrophic factor
(GDNF), highly conserved neurotrophic factor that potently promotes
the survival of many types of neurons.
[0158] Parkinson's disease (PD) is a neurodegenerative disorder
characterized by the loss of the nigrostriatal pathway; a
progressive disorder resulting from degeneration of dopaminergic
neurons within the substantia nigra. Although the cause of
Parkinson's disease is not known, it is associated with the
progressive death of dopaminergic (tyrosine hydroxylase (TH)
positive) mesencephalic neurons, inducing motor impairment. The
characteristic symptoms of Parkinson's disease appear when up to
70% of TH-positive nigrostriatal neurons have degenerated. Surgical
therapies aimed at replacing lost dopaminergic neurons or
disrupting aberrant basal ganglia circuitry have recently been
tested (C. Honey et al. 1999). However, these clinical trials have
focused on patients with advanced disease, and the primary goal of
forestalling disease progression in newly diagnosed patients has
yet to be realized. The administration can be by stereotaxic
injection. The administration can be intracranially, e.g.,
intracranially to stiatum or to substantia nigra. The
administration can also be by retrograde transport.
[0159] In an embodiment, the administration site is the striatum of
the brain, in particular the caudate putamen. Injection into the
putamen can label target sites located in various distant regions
of the brain, for example, the globus pallidus, amygdala,
subthalamic nucleus or the substantia nigra. Transduction of cells
in the pallidus commonly causes retrograde labelling of cells in
the thalamus. In a preferred embodiment the (or one of the) target
site(s) is the substantia nigra.
[0160] In another embodiment the vector system is injected directly
into the spinal cord. This administration site accesses distal
connections in the brain stem and cortex. Within a given target
site, the vector system may transduce a target cell. The target
cell may be a cell found in nervous tissue, such as a neuron,
astrocyte, oligodendrocyte, microglia or ependymal cell. In a
preferred embodiment, the target cell is a neuron, in particular a
TH positive neuron.
[0161] The vector system can be administered by direct injection.
Methods for injection into the brain (in particular the striatum)
are well known in the art (Bilang-Bleuel et al (1997) Proc. Acad.
Natl. Sci. USA 94:8818-8823; Choi-Lundberg et al (1998) Exp.
Neurol. 154:261-275; Choi-Lundberg et al (1997) Science
275:838-841; and Mandel et al (1997)) Proc. Acad. Natl. Sci. USA
94:14083-14088). Stereotaxic injections may be given.
[0162] Administration of the cells or virus into selected regions
of the recipient subject's brain may be made by drilling a hole and
piercing the dura to permit the needle of a microsyringe to be
inserted. The cells or recombinant lentivirus can alternatively be
injected intrathecally into the spinal cord region. A cell
preparation infected ex vivo, or the recombinant lentivirus of the
invention, permits grafting of neuronal cells to any predetermined
site in the brain or spinal cord, and allows multiple grafting
simultaneously in several different sites using the same cell
suspension or viral suspension and permits mixtures of cells from
different anatomical regions.
[0163] For transduction in tissues such as the brain, it is
necessary to use very small volumes, so the viral preparation is
concentrated by ultracentrifugation. The resulting preparation
should have at least 10.sup.8 pfu./ml, preferably from 10.sup.8 to
10.sup.10 pfu./ml, more preferably at least 10.sup.9 pfu./ml. (The
titer is expressed in transducing units per ml (pfu./ml) as titred
on a standard D17 cell line). It has been found that improved
dispersion of transgene expression can be obtained by increasing
the number of injection sites and decreasing the rate of injection
(Horellou and Mallet (1997) as above). Usually between 1 and 10
injection sites are used, more commonly between 2 and 6. For a dose
comprising 1-5.times.10.sup.9 pfu./ml, the rate of injection is
commonly between 0.1 and 10 .mu.l/min, usually about 1
.mu.l/min.
[0164] In another embodiment the vector system is administered to a
peripheral administration site. The vector may be administered to
any part of the body from which it can travel to the target site by
retrograde transport. In other words the vector may be administered
to any part of the body to which a neuron within the target site
projects.
[0165] The "periphery" can be considered to be all part of the body
other than the CNS (brain and spinal cord). In particular,
peripheral sites are those which are distant to the CNS. Sensory
neurons may be accessed by administration to any tissue which is
innervated by the neuron. In particular this includes the skin,
muscles and the sciatic nerve.
[0166] In another embodiment the vector system is administered
intramuscularly. In this way, the system can access a distant
target site via the neurons which innervate the innoculated muscle.
The vector system may thus be used to access the CNS (in particular
the spinal cord), obviating the need for direct injection into this
tissue. There is thus provided a non-invasive method for
transducing a neuron within the CNS. Muscular administration also
enables multiple doses to be administered over a prolonged
period.
[0167] Other neuronal disorders that can be treated similarly by
the method of the invention include Alzheimer's disease,
Huntington's disease, neuronal damage due to stroke, and damage in
the spinal cord. Alzheimer's disease is characterized by
degeneration of the cholinergic neurons of the basal forebrain. The
neurotransmitter for these neurons is acetylcholine, which is
necessary for their survival. Engraftment of cholinergic cells
infected with a recombinant lentivirus of the invention containing
an exogenous gene for a factor which would promote survival of
these neurons can be accomplished by the method of the invention,
as described. Following a stroke, there is selective loss of cells
in the CA1 of the hippocampus as well as cortical cell loss which
may underlie cognitive function and memory loss in these patients.
Once identified, molecules responsible for CA1 cell death can be
inhibited by the methods of this invention. For example, antisense
sequences, or a gene encoding an antagonist can be transferred to a
neuronal cell and implanted into the hippocampal region of the
brain.
[0168] The method of transferring nucleic acid also contemplates
the grafting of neuroblasts in combination with other therapeutic
procedures useful in the treatment of disorders of the CNS. For
example, the lentiviral infected cells can be co-administered with
agents such as growth factors, gangliosides, antibiotics,
neurotransmitters, neurohormones, toxins, neurite promoting
molecules and antimetabolites and precursors of these molecules
such as the precursor of dopamine, L-DOPA.
[0169] Further, there are a number of inherited neurologic diseases
in which defective genes may be replaced including: lysosomal
storage diseases such as those involving .beta.-hexosamimidase or
glucocerebrosidase; deficiencies in hypoxanthine phosphoribosyl
transferase activity (the "Lesch-Nyhan" syndrome"); amyloid
polyneuropathies (-prealbumin); Duchenne's muscular dystrophy, and
retinoblastoma, for example.
[0170] For diseases due to deficiency of a protein product, gene
transfer could introduce a normal gene into the affected tissues
for replacement therapy, as well as to create animal models for the
disease using antisense mutations. For example, it may be desirable
to insert a Factor IX encoding nucleic acid into a lentivirus for
infection of a muscle or liver cell.
[0171] Stem cell therapy contemplates injection of stem cells
transduced by a lentiviral vector carrying a therapeutic gene of
interest into a fetus central nervous system. The correction or
rescue of a genetic defect is achieved during cell differentiation.
Stem cells at a nondividing stage should be efficiently transduced
by such a vector using a convenient infection technique.
[0172] V. Pharmaceutical Compositions
[0173] The invention further provides pharmaceutical compositions
comprising lentiviral vectors of the invention and one or more
pharmaceutically acceptable carriers.
[0174] The pharmacologically active compounds of this invention can
be processed in accordance with conventional methods of galenic
pharmacy to produce medicinal agents for administration to
patients, e.g., mammals including humans.
[0175] The compounds of this invention can be employed in admixture
with conventional excipients, i.e., pharmaceutically acceptable
organic or inorganic carrier substances suitable for parenteral,
enteral (e.g., oral) or topical application, which do not
deleteriously react with the active compounds. Suitable
pharmaceutically acceptable carriers include but are not limited to
water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl
alcohols, polyethylene glycols, gelatin, carbohydrates such as
lactose, amylose or starch, magnesium stearate, talc, silicic acid,
viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, pentaerythritol fatty acid esters, hydroxy
methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical
preparations can be sterilized and if desired mixed with auxiliary
agents, e.g., lubricants, preservatives, stabilizers, wetting
agents, emulsifiers, salts for influencing osmotic pressure,
buffers, coloring, flavoring and/or aromatic substances and the
like which do not deleteriously react with the active compounds.
They can also be combined where desired with other active agents,
e.g., vitamins.
[0176] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. Ampoules are convenient unit dosages.
[0177] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules. A syrup,
elixir, or the like can be used wherein a sweetened vehicle is
employed.
[0178] Sustained or directed release compositions can be
formulated, e.g., by inclusion in liposomes or those wherein the
active compound is protected with differentially degradable
coatings, e.g., by microencapsulation, multiple coatings, etc. It
is also possible to freeze-dry these compounds and use the
lyophilizates obtained, for example, for the preparation of
products for injection.
[0179] For topical application, there are employed as non-sprayable
forms, viscous to semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include but
are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, aerosols, etc., which are,
if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives, stabilizers, wetting agents, buffers or salts for
influencing osmotic pressure, etc. For topical application, also
suitable are sprayable aerosol preparations wherein the active
ingredient, preferably in combination with a solid or liquid inert
carrier material, is packaged in a squeeze bottle or in admixture
with a pressurized volatile, normally gaseous propellant, e.g., a
freon.
[0180] It will be appreciated that the actual preferred amounts of
active compound in a specific case will vary according to the
specific compound being utilized, the compositions formulated, the
mode of application, and the particular situs and organism being
treated. Dosages for a given host can be determined using
conventional considerations, e.g., by customary comparison of the
differential activities of the subject compounds and of a known
agent, e.g., by means of an appropriate, conventional
pharmacological protocol.
[0181] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0182] The lentiviral vectors of the invention can also be
administered in combination with other agents, for example,
chemotherapeutic agents, radiation treatment, or steroids,
according to methods known and described in the art. PCT
Publication WO 2008/08069942, "Novel Methods of Enhancing Delivery
of a Gene Therapy Vector Using Steroids," describes methods for
enhancing expression of a viral vector-encoded therapeutic gene
product by delivering to a subject the viral vector in conjunction
with a steroid, e.g., prednisolone, cortisone, corticosterone, or
dexamethasone. In embodiments, the individual therapies being
combined are not necessarily administered together, e.g., they can
be administered separately via different modes of administration,
alternately, etc.
[0183] VII. Patients
[0184] The invention contemplates treatment of patients including
human patients. The term patient as used in the present application
refers to all different types of mammals including humans and the
present invention is effective with respect to all such mammals.
The present invention is effective in treating any mammalian
species which have a disease potentially remedied by delivery of a
gene product or inhibition of expression of a gene.
[0185] The contents of all cited references, including literature
references, issued patents, published patent applications, and
co-pending patent applications, cited throughout this application
are hereby expressly incorporated by reference in their
entirety.
[0186] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims. The
following examples are offered by way of illustration of the
present invention, and not by way of limitation.
EXAMPLES
Example 1
Vector Construction
[0187] The additional components of the gene transfer system
include a packaging (helper) plasmid and an envelope (Env) plasmid
encoding VSV-G driven by the HIV-1 LTR (Mochizuki, H. et al., 1998,
J Virol 72:8873-8883; Reiser, J. et al., 1996, PNAS USA
93:15266-15271). These packaging and envelope constructs are
described herein and by Lai et al., 2000, PNAS, 97: 11297-11302,
and Lai, et al., 2002, Neurosci. Res. 67: 363-371, incorporated
herein by reference. The helper construct has a deletion in the
packaging signal rendering it inactive, and the 5' LTR is replaced
with the CMV-IE promoter. The CMV promoter was derived from pcDNA
3.1, which was used as a template for a PCR reaction yielding 590
by of the CMV promoter. The HIV-1 helper construct was digested by
EcoRV (33) and Afl II (517) to have generate a deletion of 475 by
from the U3 region of the 5'LTR. Insertion of the CMV promoter PCR
fragment was then ligated into the HIV-1 helper construct to create
a safer Tat-independent construct without compromising viral titer.
The viral genes tat and nef were also inactivated. Pseudotyped
vectors were produced in human embryonic kidney 293T cells using a
three-component transient packaging system (Mochizuki, H. et al.,
1998).
[0188] The transfer vectors were based on HIV-1 lentivirus vectors,
and were made using methods similar to those described by Kim, et
al., J. Gene Med. (2005), referenced above. The transfer vectors of
the present invention are self-inactivating, i.e., they have a
deletion in the U3 region of the 5' LTR that was introduced as
follows: (a) the fragment between Nef and the 3' LTR was isolated
by digesting with Xho-I and Afl-II; (b) the isolated fragment of
about 820 by was subcloned into the PUC18 vector; (c) the U3 region
(containing the TATA box, SP1) of the 3' LTR of about 337 by was
deleted by EcoR-V and Rsa-I; and (d) a PCR fragment from (c)
containing the regions of Nef and the 3' LTR with the U3 deletion
were ligated back into the lentiviral vector.
Example 2
In Vitro Infection of a Prostate Cancer Cell Line with
SIN-HIV-P53-EGFP
[0189] PC-3 (prostate cancer) cells were infected with a lentivirus
containing a gene transfer vector expressing wild-type P53 and
EGFP. Three groups were tested. Treatment group: PC-3 cells were
treated with SIN-HIV-P53-EGFP (construct shown in FIG. 6). Control
(negative) group: PC-3 cells were untreated. Control (positive)
group: Normal cells were treated or not treated with
SIN-HIV-P53-EGFP.
[0190] Prostate cancer cell-lines (PC-3) and 293-T cells were
placed into the 12 well-plate with 0.4.times.10.sup.6 cells, and
were cultured in the RPMI 1640 and DMEM medium containing
antibiotics. 200 .mu.l of the packaged lentiviral vector
SIN-CMV-p53-IRES/CS-EGFP (see FIG. 1C) was added to the cells for
the infection study under 1 mg/ml. The cells were then cultured by
incubating for 6-16 hours at 37.degree. C. in a CO.sub.2 incubator.
After infection for 48-72 hours, we evaluated the expression of
EGFP using a fluorescence microscope in both the transduced PC-3
and 239-T cells. We found that a high level of EGFP was expressed
in both PC-3 and 293-T cells transduced by
SIN-CMV-p53-IRES/CS-EGFP, but not in the control group of those
cells that were not infected (FIGS. 8A-C). The results indicated
that our vector system worked well to express both transgenes
simultaneously.
[0191] After treatment, RNA was isolated from the cells, and RT-PCR
was used to determine the p53 expression level at different time
points. Corresponding cell growth curves and survival cell numbers
were determined at different time points, and the results for
treated and untreated cells compared.
[0192] To evaluate confirm P53 expression, we extracted total RNA
96 hours after infection. 0.5 .mu.g total RNA from each group
(treated and untreated) were used as template for RT-PCR. P53 mRNA
was amplified using sense primer (from the partial promoter of CMV:
5'-tacgtattagtcatcgctatt-3) and antisense primer (from the end of
the P53 gene: 5'-aggcctcattcagctctcgga-3'). The results showed that
the cell lines infected by the vector expressed a high level of
P53, and the uninfected cell lines expressed only the basic level
of endogenous p53 (see FIG. 8D). Thus, our data indicated that our
vector system is functional for highly expressing both the two
transgene protein in the cells at the same time.
[0193] We also compared growth and survival of the prostate cancer
cells transduced with the vector with untreated cells. Taking time
points for up to one week, we observed the growth condition and
cell numbers under the light and fluorescent microscopes.
Expression of EGFP indicated transduction by the vector. We
observed that PC-3 cells transduced by the vector died quickly
compared with the untreated PC-3 cells, and that both treated and
untreated 293-T cells showed growth and no significant cell death.
The result can be confirmed using FACS analysis to quantitate cell
numbers at the respective time points.
Example 3
In Vivo Transduction with SIN-HIV-P53-EGFP and SIN-HIV-P53-Bcl-2
RNAi
[0194] Transduction with the SIN-HIV-P53-EGFP vector described in
Example II, expressing P53 and GFP, or SIN-HIV-P53-Bcl-2 RNAi,
expressing P53 and an RNAi agent targeting human Bcl-2, is done in
vivo. See FIG. 1C, Panel B, upper construct (CMV-P53-hPSA-Bcl-2
RNAi). The NOD SCID mouse model, characterized by a major
immunodeficiency, is used to study gene therapy for prostate cancer
using lentiviral transfer systems of the invention.
[0195] NOD SCID mice are subcutaneously implanted with a PC-3 cell
suspension in a thoracic postero-lateral wound. Tumor cell
suspensions are injected using a 30-gauge needle and a 1-ml
disposable syringe. The volume of inoculation is 100 .mu.l
(2.times.10.sup.6 tumor cells suspended in 100 .mu.l of PBS). After
tumor appearance (1-2 weeks post-implantation), the virus
injections are made.
[0196] Tumor progression is monitored by palpation twice a week by
the investigator, and subcutaneous tumor size is measured using a
caliper. Viral vector is administered at a titer of about 10.sup.8
pfu/ml, by tail-vein injection. Animals are euthanized according to
tumor size or clinical status during the observation period.
Cervical dislocation is performed 2 to 4 weeks after injection.
[0197] Prostate cancer tumor sizes are measured, and for EGFP
immunostaining to evaluate distribution of the vector, liver, lung,
heart, and bone marrow cells are harvested. These tissue samples
are collected for immunostaining does as described by Lai, et al.,
PNAS, 2002. The tissues are subjected to total RNA and protein
extractions, to evaluate expression of the transgenes. The tissue
slides are made and examined for EGFP fluorescence under the
fluorescence microscope, to determine the distribution of the
vector and thereby identify target tissues in the animal model. P53
expression is evaluated by RT-PCR of tissue observed to express
EGFP under the fluorescent microscope. The function of the RNAi
agent targeted to bcl-2 is be tested by both RT-PCR of RNA and
Western-blot protein analysis in the same samples, to determine
whether the level of Bcl-2 in tumor tissue is significantly
downregulated in the same target cells that express EGFP.
[0198] Co-localization of the target cells expressing the P53 or
RNAi and EGFP simultaneously is evaluated, particularly those cells
in tumors that underwent size reduction as a result of vector
treatment. The distribution, e.g., in bone marrow, of the vector
after i.v. injection is also determined, to provide information
useful for human clinical trials using the vector coexpressing P53
and a bcl-2 RNAi agent.
Example 4
In Vitro Synergistic Effects of SIN-HIV-P53-Bcl-2 RNAi
[0199] PC3 prostate cancer cells grown on a substrate were
contacted with double gene viral vector construct that express P53
and Bcl-2 RNAi, which is discussed above. FIGS. 10A and 10B show in
vitro test results that show that double gene (P53 and Bcl-2 RNAi)
expression construct viral vector induces cell necrosis of PC3
prostate cancer cells. In living culture, FIG. 10A shows the
untreated cells, and the FIG. 10B shows cells treated with the
double gene vector.
[0200] However, when tested on human embryonic kidney cells (293T),
the double gene construct did not cause necrosis. FIG. 11A shows
the untreated cells and FIG. 11B shows cells treated with the viral
vector construct P53-Bcl-2 RNAi, expressing P53 and an RNAi agent
targeting human Bcl-2. No difference in necrosis is observed,
indicating specificity of the double gene construct for prostate
tumor. The cells were observed three days after infection.
Example 5
In Vivo Synergistic Effects of SIN-HIV-P53-Bcl-2 RNAi Over P53 and
Bcl RNAi Alone--First Study
[0201] In vivo effects of the double gene construct and the
individual single gene constructs were compared against prostate
tumor. The results show that synergistic tumor reducing effects
were seen for the viral vector construct P53-Bcl-2 RNAi, expressing
P53 and an RNAi agent targeting human Bcl-2. FIG. 12 is a graph
showing this effect. In order from left to right, far left bar is
untreated control group; P53 alone expressed through viral vector
construct (repair); Bcl-2 siRNA alone expressed through viral
vector construct (downregulation of BCL2 gene) only; and on the far
right viral vector construct P53-Bcl-2 RNAi, expressing P53 and an
RNAi agent targeting human Bcl-2 for simultaneous P53 (repair) and
Bcl-2 siRNA. Whereas application of P53 gene construct alone
reduced the tumor weight to 1.71 grams, and 1.5 grams when only
Bcl-2 siRNA construct was applied, application of the combination
gene construct resulted in the tumor weight of 0.71 grams,
indicating a synergistic effect of the double gene construct.
[0202] In the in vivo studies, 100 .mu.l of viruses
(5.0.times.10.sup.8 cells) were injected into the tail vein of mice
for three weeks. After injection for three weeks, the mice were
sacrificed and tumors were harvested and weighed.
[0203] Further, cells obtained from the untreated tumor and tumor
treated with the double gene contruct were examined for human
prostate-specific antigen (hPSA) expression. FIGS. 13A and 13B show
staining of control (untreated tumor) versus the treatment group.
The results show that tumor cells treated with viral vector
construct P53-Bcl-2 RNAi, expressing P53 and an RNAi agent
targeting human Bcl-2 no longer produce hPSA, which means that the
cells are no longer active tumor cells or are no longer actively
cancerous. After human prostate specific antibody staining, FIG.
13A shows the control tumor, which expresses hPSA, and FIG. 13B
shows treatment tumor that shows cell necrosis and no expression of
hPSA.
Example 6
In Vivo Synergistic Effects of SIN-HIV-P53-Bcl-2 RNAi Over P53 and
Bcl RNAi Alone--Second Study
[0204] A second study was carried out using mouse models for
prostate cancer using the individual gene constructs for P53 and
Bcl-2 RNAi, and the double gene construct that carry both of these
genes. Multiple mice were tested. V1 Group is mice treated with P53
gene construct alone, V2 Group is mice treated with Bcl-2 RNAi
construct alone, and V3 Group is mice treated with a double gene
construct expressing both P53 and Bcl-2. PC is the untreated
control mice. As shown in the table in FIGS. 14A-14C, the mice were
injected with the constructs and their tumor size measured.
Injection days were Days 0, 5, 9, 14 and 21. Tumor size measurement
days were Days 7, 12, 16, 21 and 28. Comparison of the tumor size
of the V1, V2, V3 mice show that the tumor size in these mice were
much reduced compared with the tumor size in the control PC mice.
Comparison of the tumor weight on Day 28 in FIG. 14C indicates that
V1, V2, V3 mice tumor weight were much reduced compared with the
tumor weight in the control PC mice. The ratio of tumor
weight/starting tumor size at day 7 shows a large difference in the
V1, V2 and V3 Groups compared with that of the PC control mice.
[0205] V3 Group was injected with half of the dosage concentration
compared with V1 and V2 Groups. Yet its tumor size is smaller than
V2 and close to V1, thus indicating the synergistic effect of
V3.
Example 7
Treatment of Human Patients with
SIN-HIV-CMV-p53-hPSA-RNAi-huCD25
[0206] To investigate the combination gene therapy effects of our
gene vector for treatment of human metastatic prostate cancer,
adult bone marrow cells (BMC) genetically modified by transduction
with SIN-HIV-CMV-p53-hPSA-RNAi-huCD25 (FIG. 1C, Panel C, lower
construct) are administered to patients with metastasized prostate
cancer by the following procedure: (1) blood is harvested from the
patient to collect stem cells. Recent medical advances now make it
possible to collect stem cells from circulating blood as well. The
collection or harvesting of bone marrow is typically done in a
hospital operating room under general anesthesia. The bone marrow
is then frozen and stored until gene therapy is completed. (2) The
bone marrow cells are transduced ex vivo by the gene transfer
vector expressing P53, RNAi and huCD25. To select only those cells
stems transduced by the transfer vector, cells expressing the
selectable marker huCD25 are selected. Stem cell selection is one
method used for purging tumor cells. Recent clinical studies have
demonstrated that stem cell selection reduces the tumor
contamination found in mobilized blood. This is possibly because
stem cells have unique properties not shared by tumor cells. (3)
After evaluation of the transduced stem cells, and marker
selection, the stem cells are thawed and returned to the patient.
This procedure is often referred to as the transplant. Within a few
days after completing the gene therapy, the stored stem cells are
transplanted, or re-infused into the patient's bloodstream. The
re-infusion process is similar to a blood transfusion and takes
place in the patient's room: it is not a surgical procedure. The
frozen bags of bone marrow or blood cells are thawed in a warm
water bath, and then injected into the bloodstream through the
catheter. It usually takes 2-4 hours for the infusion. Infused stem
cells travel through the bloodstream, and eventually, to the bone
marrow where they begin to produce new white blood cells, red blood
cells, and platelets. (3) The patients are evaluated by collection
of their blood to determine the level of P53, Bcl-2, and the
selectable marker (i.e., huCD25) at different time points following
treatment using Real Time PCR. At the same time a routine diagnosis
of index is done to see if any indexes of cancer markers disappear
in the current clinical settings.
[0207] All of the references cited herein are incorporated by
reference in their entirety.
[0208] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention
specifically described herein. Such equivalents are intended to be
encompassed in the scope of the claims.
Sequence CWU 1
1
1415PRTArtificial Sequencetransport motif 1Arg Ser Ala Ser Arg1
525PRTArtificial Sequencetransport motif 2Arg Thr Ala Ser Arg1
535PRTArtificial Sequencetransport motif 3Arg Ser Arg Ala Arg1
545PRTArtificial Sequencetransport motif 4Arg Thr Arg Ala Arg1
555PRTArtificial Sequencetransport motif 5Ala Thr Ala Thr Arg1
566PRTArtificial Sequencetransport motif 6Arg Ser Ala Ala Ser Arg1
579PRTArtificial Sequencetat derived peptide 7Gly Arg Lys Lys Arg
Arg Gln Arg Arg1 5833DNAArtificial Sequencenucleic acid encoding
tat derived peptide 8tatggcagga agaagcggag acagcgacga aga
33911PRTArtificial Sequencetat derived peptide 9Tyr Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg1 5 101011PRTArtificial Sequencetat derived
peptide 10Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala1 5
101111PRTArtificial Sequencetat derived peptide 11Thr His Arg Leu
Pro Arg Arg Arg Arg Arg Arg1 5 101211PRTArtificial Sequencetat
derived peptide 12Gly Gly Arg Arg Ala Arg Arg Arg Arg Arg Arg1 5
101324DNAArtificial SequencePrimer 13agctagatag tgtcacctaa atgc
241415DNAArtificial SequencePrimer 14agcatgcctg ctatt 15
* * * * *