U.S. patent application number 11/446353 was filed with the patent office on 2007-01-25 for method of targeted gene delivery using viral vectors.
Invention is credited to David Baltimore, Ping Wang, Lili Yang.
Application Number | 20070020238 11/446353 |
Document ID | / |
Family ID | 37482360 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070020238 |
Kind Code |
A1 |
Baltimore; David ; et
al. |
January 25, 2007 |
Method of targeted gene delivery using viral vectors
Abstract
Methods and compositions are provided for delivering a
polynucleotide encoding a gene of interest to a target cell using a
virus. The virus envelope comprises a cell-specific binding
determinant that recognizes and binds to a component on the target
cell surface, leading to endocytosis of the virus. A separate
fusogenic molecule is also present on the envelope and facilitates
delivery of the polynucleotide across the membrane and into the
cytosol of the target cell. The methods and related compositions
can be used for treating patients having suffering from a wide
range of conditions, including infection, such as HIV; cancers,
such as non-Hodgkin's lymphoma and breast cancer; and hematological
disorders, such as severe combined immunodeficiency.
Inventors: |
Baltimore; David; (Pasadena,
CA) ; Wang; Ping; (Pasadena, CA) ; Yang;
Lili; (Pasadena, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
37482360 |
Appl. No.: |
11/446353 |
Filed: |
June 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60686215 |
Jun 1, 2005 |
|
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60738078 |
Nov 19, 2005 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456; 435/5; 514/44A; 977/802 |
Current CPC
Class: |
A61K 2039/505 20130101;
C12N 15/86 20130101; C07K 2319/00 20130101; C12N 2740/16043
20130101; C07K 2317/53 20130101; C07K 16/2887 20130101; C12N
2740/15043 20130101; Y02A 50/30 20180101; C12N 2799/027 20130101;
C07K 2319/03 20130101; C12N 2810/851 20130101; C12N 2810/859
20130101; Y02A 50/396 20180101; C12N 2740/16045 20130101; C12N
2810/80 20130101; Y02A 50/386 20180101; C07K 2317/622 20130101 |
Class at
Publication: |
424/093.2 ;
514/044; 435/456; 435/005; 435/235.1; 977/802 |
International
Class: |
A61K 48/00 20070101
A61K048/00; C12N 15/867 20070101 C12N015/867; C12Q 1/70 20060101
C12Q001/70; C12N 7/00 20060101 C12N007/00 |
Claims
1. A method of delivering a polynucleotide to a target cell, the
method comprising: infecting the target cell with a recombinant
retrovirus, wherein the recombinant retrovirus comprises the
polynucleotide to be delivered, the R and U5 sequences from a 5'
lentiviral long terminal repeat (LTR), a self-inactivating
lentiviral 3' LTR, a fusogenic molecule, and a cell-specific
binding determinant.
2. The method of claim 1, wherein the fusogenic molecule is pH
sensitive.
3. The method of claim 1, wherein the fusogenic molecule is a class
I fusogen.
4. The method of claim 1, wherein the fusogenic molecule is a class
II fusogen.
5. The method of claim 1, wherein the fusogenic molecule is viral
glycoprotein.
6. The method of claim 5, wherein the fusogenic molecule has a
reduced binding ability.
7. The method of claim 5, wherein the fusogenic molecule comprises
a viral glycoprotein derived from one of the group of viruses
consisting of: Lassa fever virus, tick-borne encephalitis virus,
Dengue virus, Hepatitis B virus, Rabies virus, Semliki Forest
virus, Ross River virus, Aura virus, Borna disease virus, Hantaan
virus, and SARS-CoV virus.
8. The method of claim 1, wherein the cell-specific binding
determinant comprises a protein.
9. The method of claim 1, wherein the cell-specific binding
determinant comprises an antibody.
10. The method of claim 9, wherein the antibody comprises the light
and heavy constant chain regions of human IgG1.
11. The method of claim 11, wherein the cell-specific binding
determinant further comprises immunoglobulin alpha and
immunoglobulin beta.
12. The method of claim 9, wherein the antibody is a single chain
antibody.
13. The method of claim 12, wherein the single chain antibody is
fused with a transmembrane domain from another protein.
14. The method of claim 9, wherein the antibody is selected from
the group consisting of an anti-CD20 antibody, an anti-CD34
antibody and an anti-DEC-205 antibody.
15. The method of claim 1, wherein the 5' LTR sequences are from
HIV.
16. The method of claim 1 wherein the self-inactivating 3' LTR
comprises a U3 element with a deletion of its enhancer
sequence.
17. The method of claim 16, wherein the self-inactivating 3' LTR is
a modified HIV 3' LTR.
18. The method of claim 1, wherein the recombinant retrovirus is
pseudotyped.
19. The method of claim 1, wherein the polynucleotide is a gene of
interest.
20. The method of claim 1, wherein the target cell is a cancer
cell.
21. The method of claim 1, further comprising the steps of:
transfecting a packaging cell line with a vector comprising the
polynucleotide to be delivered, the R and U5 sequences and the
self-inactivating lentiviral 3' LTR, a vector comprising a gene
encoding the fusogenic molecule, and a vector comprising a gene
encoding the cell-specific binding determinant; and recovering
recombinant retrovirus from the packaging cell line.
22. The method of claim 21, wherein said packaging cell line is a
293 cell line.
23. A recombinant retrovirus comprising: a nucleic acid having the
R and U5 sequences from a 5' lentiviral long terminal repeat (LTR);
a self-inactivating lentiviral 3' LTR; a a fusogenic molecule, and
a membrane-bound cell-specific binding determinant.
24. The recombinant retrovirus of claim 23, wherein the fusogenic
molecule is a viral glycoprotein.
25. The recombinant retrovirus of claim 23, wherein the fusogenic
molecule is pH sensitive.
26. The recombinant retrovirus of claim 23, wherein the fusogenic
molecule is mutant hemagglutinin.
27. The recombinant retrovirus of claim 23, wherein the fusogenic
molecule is SIN.
28. The recombinant retrovirus of claim 23, wherein the
cell-specific binding determinant comprises a protein.
29. The recombinant retrovirus of claim 23, wherein the
cell-specific binding determinant comprises an antibody.
30. The recombinant retrovirus of claim 29, wherein the antibody is
human IgG.sub.1.
31. The recombinant retrovirus of claim 30, wherein the
cell-specific binding determinant further comprises immunoglobulin
alpha and immunoglobulin beta.
32. The recombinant retrovirus of claim 29, wherein the antibody is
a single chain antibody.
33. The recombinant retrovirus of claim 29, wherein the viral
construct further comprises at least one gene of interest.
34. The recombinant retrovirus of claim 33, wherein the gene of
interest is a reporter gene.
35. The method of claim 33, wherein the gene of interest encodes an
siRNA molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn. 119(e) to U.S. Provisional Application No. 60/686,215, filed
Jun. 1, 2005 and U.S. Provisional Application No. 60/738,078, filed
Nov. 19, 2005, which are both herein expressly incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates generally to targeted gene delivery,
and more particularly to the use of a recombinant virus comprising
a fusogenic molecule and a distinct affinity molecule.
[0004] 2. Description of the Related Art
[0005] The delivery of functional genes and other polynucleotides
into particular target cells can be used in a variety of contexts.
For example, gene therapy can be used to prevent or treat disease.
A particularly desirable gene delivery protocol would be able to
precisely deliver a gene of interest to specific cells or organs in
vivo. Certain viruses are naturally suited for gene delivery, and
significant effort has been focused on engineering viral vectors as
gene transfer vehicles. Among these viruses, oncoretroviral and
lentiviral vectors exhibit promising features because they have the
ability to produce stable transduction, maintain long-term
transgene expression and, for lentiviruses, enable transduction of
non-dividing cells. Targeting such viruses to particular cell types
has proved to be challenging.
[0006] Many attempts have been made to develop targetable
transduction systems using retroviral and lentiviral vectors (see,
for example, D. Lavillette, S. J. Russell, F. L. Cosset, Curr.
Opin. Biotech. 12, 461 (2001), V. Sandrin, S. J. Russell, F. L.
Cosset, Curr. Top. Microbio. Immunol. 281, 137 (2003)). Significant
effort has been devoted to altering the envelope glycoprotein
(env), the protein that is responsible for binding the virus to
cell surface receptors and for mediating entry. The plasticity of
the surface domain of env allows insertion of ligands, peptides and
single-chain antibodies, which can direct the vectors to specific
cell types (N. V. Somia, M. Zoppe, I. M. Verma, Proc. Natl. Acad.
Sci. USA 92, 7570 (1995)). However, this manipulation adversely
affects the fusion domain of env, resulting in low viral titers.
The unknown and delicate coupling mechanisms of binding and fusion
make it extremely difficult to reconstitute fusion function once
the surface domain of the same molecule has been altered.
[0007] Another approach involves complex env with a ligand protein
or antibody to form a bridge to attach the virus to specific cells
(e.g., K. Morizono, G. Bristol, Y. M. Xie, S. K. P. Kung, I. S. Y.
Chen, J. Virol. 75, 8016 (2001). The challenge to this approach is
that env, once complexed with the one end of the bridge molecule,
fuses inefficiently. Since no practical strategies are available
for targeted in vivo gene delivery, current gene therapy clinical
trails are generally based on in vitro transduction of purified
cells followed by infusion of the modified cells into the patient.
This in vitro approach is an expensive procedure with significant
safety challenges.
SUMMARY OF THE INVENTION
[0008] According to one aspect of the invention, methods are
provided for delivering a polynucleotide to a desired cell,
comprising infecting a target cell with a recombinant retrovirus.
In other aspects, recombinant retrovirus and various vectors and
constructs for producing the recombinant retrovirus are
provided.
[0009] The envelope of the recombinant retrovirus preferably
comprises a fusogenic molecule and a cell-specific binding
determinant. In some embodiments, the recombinant retrovirus
preferably comprises the R and U5 sequences from a 5' lentiviral
long terminal repeat (LTR), a self-inactivating lentiviral 3' LTR,
a fusogenic molecule, and a cell-specific binding determinant. In
some embodiments, the recombinant retrovirus is produced from the
FUGW construct.
[0010] The fusogenic molecule is preferably a viral glycoprotein
and may be, for example, a class I or class II fusogen. In some
embodiments of the invention, the fusogenic molecule is pH
sensitive. Preferably the pH sensitivity is such that the fusogen
is able to mediate delivery of the viral core across the membrane
in the endocytic compartment of a target cell. In some embodiments,
the fusogenic molecule may have a lowered affinity for a cognate
molecule, such as a receptor or antigen.
[0011] In some embodiments, the fusogenic molecule is
hemagglutinin, preferably a mutant hemagglutinin. In other
embodiments, the fusogenic molecule is SIN. In still other
embodiments, the fusogenic molecule comprises a viral glycoprotein
derived from one of the following viruses: Lassa fever virus,
tick-borne encephalitis virus, Dengue virus, Hepatitis B virus,
Rabies virus, Semliki Forest virus, Ross River virus, Aura virus,
Borna disease virus, Hantaan virus, and SARS-CoV virus.
[0012] In some embodiments of the invention, the cell-specific
binding determinant comprises a protein that is able to bind with
specificity to a cell surface molecule present on a target cell.
Preferably, the binding is high-affinity binding and in some
embodiments the dissociation constant may be in the range of
10.sup.-6 to 10.sup.-12 M. However, in other embodiments lower or
higher affinity binding is possible.
[0013] The cell-specific binding determinant is preferably a
protein, and in some embodiments is an antibody. The cell-specific
binding determinant may comprise more than one molecule. For
example, if the cell-specific binding determinant comprises an
antibody, it may also comprise immunoglobulin alpha and
immunoglobulin beta. If the antibody is, for example, a single
chain antibody, it may be fused with a transmembrane domain from
another protein. In some particular embodiments of the invention,
the antibody is a CD20 antibody, a CD34 antibody or an antibody
against DEC-205.
[0014] In some embodiments of the invention, the 5' LTR sequences
are from HIV. The self-inactivating 3' LTR may comprise a U3
element with a deletion of its enhancer sequence. The
self-inactivating 3' LTR may be a modified HIV 3' LTR. The
recombinant retrovirus may comprise other elements, such as the
woodchuck hepatitis virus enhancer element sequence and/or a tRNA
amber suppressor sequence.
[0015] The recombinant retrovirus may be pseudotyped, for example,
with the vesicular stomatitits virus envelope glycoprotein or
ecotropic envelope protein 4.17.
[0016] In preferred embodiments, the recombinant retrovirus further
comprises one or more genes of interest to be delivered to the
target cell. The gene of interest is not limited in any way, and
may, for example, encode a protein to be expressed in the cell. In
other embodiments the gene of interest encodes an siRNA or other
molecule to be expressed in the cell. In some embodiments at least
one of the genes of interest is a reporter gene, such as a
fluorescent protein. In some embodiments, the fluorescent protein
is green fluorescent protein.
[0017] The gene of interest is preferably linked to a promoter,
such as an RNA Polymerase II or a Polymerase III promoter. In some
embodiments, the promoter is a ubiquitous promoter. For example,
the ubiquitous promoter may be selected from the group consisting
of the ubiquitin promoter, the CMV .beta.-actin promoter and the
pgk promoter. In other embodiments the promoter may be a tissue
specific promoter. A tissue specific promoter, if present, may, for
example, be selected from the group consisting of the lck promoter,
the myogenin promoter and the thy1 promoter.
[0018] The recombinant retrovirus may additionally comprise an
enhancer operably linked to the promoter. In some embodiments, the
enhancer and promoter are both CMV sequences.
[0019] In some embodiments of the invention, a packaging cell line
is transfected with one or more vectors encoding the retroviral
elements, the gene of interest, the fusogenic molecule and the
cell-specific binding determinant. Recombinant retrovirus is
collected from the packaging cell line and used to infect target
cells, thereby delivering the gene of interest to the target cells.
In some embodiments of the invention, the packaging cell line is a
293 cell line.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a schematic representation of the generation of
recombinant virus bearing a cell-specific binding determinant and a
fusogenic molecule according to some embodiments of the
invention.
[0021] FIG. 2 illustrates a molecular mechanism of targeted
transduction of a viral particle that includes a fusogenic molecule
and an affinity molecule.
[0022] FIG. 3A illustrates the fusion protein HAmu derived from
influenza A (FPV) hemagglutinin (HA). HA contains two glycoproteins
after maturation: HA1 for binding to cell surface receptor, sialic
acid; and HA2 for triggering membrane fusion. Three point mutations
within the receptor binding sites (a1: Y106F, a2: E199Q, a3: G237K)
were introduced to generate a binding-defective but
fusion-competent fusogenic molecule (HAmu).
[0023] FIG. 3B illustrates a FACS analysis of virus-producing
cells. 293T cells that were transiently transfected with separate
plasmids encoding the following: the lentiviral vector FUGW; the
membrane-bound antibody .alpha.CD20; the accessory proteins
Ig.alpha. and Ig.beta.; the fusion protein HAmu; and viral gag,
pol, and rev genes. Expression of .alpha.CD20 and HAmu was detected
using anti-human IgG antibody and anti-FPV HA antibody.
[0024] FIG. 3C illustrates the fusion protein SINmu derived from
Sindbis viral glycoprotein (SIN). SIN contains two membrane
glycoproteins (E1 and E2) and a signal peptide (E3): E1 for
mediating fusion; E2 for receptor binding; E3 as a signal sequence
for processing of E2 glycoprotein. A ten-residue detection tag
sequence was inserted between amino acid 71 and 74 of the E2
glycoprotein. A series of alterations (a4: deletion of amino acids
61-64 of E3; a5: mutation of 68SLKQ71 into 68AAAA71; ab: mutation
of 157KE158 into 157AA158) was introduced to yield the binding
defective and fusion competent SINmu fusion molecule.
[0025] FIG. 3D is similar to the FACS analysis shown in FIG. 3B,
except that SINmu was used for the fusion protein and was detected
by an anti-tag antibody.
[0026] FIG. 4A illustrates FACS analysis of target cell line
293T/CD20. CD20 expression was detected using anti-CD20 antibody.
The solid line shows expression of CD20 in 293T/CD20; the shaded
line shows CD20 expression in 293T cells (as a control).
[0027] FIG. 4B is a schematic representation of a three-staining
scheme used for analyzing virus-cell binding. Three stains were
used to detect the presence of CD20, .alpha.CD20 and the fusogenic
molecule (HAmu or SINmu), respectively.
[0028] FIG. 4C, left panel, illustrates FACS plots of 293T/CD20
cells incubated with FUGW/.alpha.CD20+HAmu. The binding of virus to
293T/CD20 cells was probed with antibody against .alpha.CD20
(anti-IgG) and HAmu. The solid line indicates analysis on
293T/CD20; the shaded line shows analysis on 293T (as a control).
The right panel of FIG. 4C shows FACS plots of 293T/CD20 cells
incubated with FUGW/aCD20+SINmu. The binding of virus to 293T/CD20
cells was detected by antibody against .alpha.CD20 and SINmu. The
solid line shows analysis on 293T/CD20; the shaded line shows
analysis on 293T (as a control).
[0029] FIG. 4D illustrates co-display of antibody and fusogenic
protein through a density plot correlating the presence of the two
proteins.
[0030] FIG. 5A shows density plots illustrating targeting of
recombinant lentivirus bearing both antibody and fusion protein to
293T/CD20 cells in vitro. 293T/CD20 cells (2.times.10.sup.5) were
transduced with 500 .mu.L fresh unconcentrated FUGW/.alpha.CD20 (no
HAmu), FUGW/HAmu (no .alpha.CD20), or FUGW/.alpha.CD20 +HAmu. 293T
cells that did not express CD20 were included as controls. The
resulting GFP expression was analyzed by FACS. The specific
transduction titer for FUGW/.alpha.CD20+HAmu was estimated to be
.about.1.times.10.sup.5 TU/mL.
[0031] FIG. 5B illustrates results from a similar transduction
experiment to that shown in FIG. 5A, but performed using
unconcentrated FUGW/SINmu (no .alpha.CD20) or
FUGW/.alpha.CD20+SINmu. For comparison of targeting specificity,
cells were also transduced with FUGW/HAmu. The specific
transduction titer for FUGW/.alpha.CD20+SINmu was estimated to be
.about.1.times.10.sup.6 TU/mL.
[0032] FIG. 5C illustrates evidence of pH-dependent fusion of HAmu
and SINmu by a cell-cell fusion assay. 293T cells
(0.1.times.10.sup.6) transiently transfected to express GFP and
surface .alpha.CD20 and fusion protein (either HAmu or SINmu), and
293T/CD20 cells were mixed together, washed once with normal PBS
(pH=7.4), and incubated in low pH PBS (pH=5.0) or normal pH PBS (as
a control) for half an hour at 37.degree. C. The cells were then
washed and cultured in the regular medium for one day. Cells were
visualized by epifluorescence microscope equipped with a GFP filter
set.
[0033] FIG. 5D illustrates the effect of addition of soluble
.alpha.CD20 on transduction with viral particles displaying
.alpha.CD20 and a fusogenic protein. .alpha.CD20 was added into
viral supernatants during transduction for 8 hours. Then the
supernatants were replaced with fresh medium. The cells were
analyzed for GFP expression after two days. Isotype-matched
antibody was used as a control.
[0034] FIG. 5E illustrates the pH dependence of transduction based
on the effect of addition of NH.sub.4Cl (instead of soluble
.alpha.CD20).
[0035] FIG. 6 shows the targeting of CD20+ human primary B cells in
vitro and in vivo using engineered lentivirus. FIG. 6A illustrates
expression of a gene of interest (GFP) in fresh, unfractionated
human PBMCs (2.times.10.sup.6) transduced by co-culturing with
concentrated FUGW/.alpha.CD20+SINmu, CCMV/.alpha.CD20+SINmu or
CPGK/.alpha.CD20+SINmu virus (2.times.10.sup.6 TU). LPS (50
.mu.g/mL) was added into the culture media for B cell survival and
growth. After two days, the B cell population was identified by
co-staining of CD19 and CD20. The solid line indicates expression
in transduced cells; the shaded line shows expression in
untransduced cells.
[0036] FIG. 6B shows stable integration of the transgene as
detected by genomic PCR amplification using a pair of GFP-specific
primers.
[0037] FIG. 6C shows expression of a gene of interest in target
cells following in vivo delivery of virus. Fresh human PBMCs were
transferred into irradiated RAG2.sup.-/-.gamma..sub.c.sup.-/- mice
(100.times.10.sup.6/mouse) via tail vein injection. Six hours
later, concentrated virus (100.times.10.sup.6 TU/mouse) was
injected through the tail vein. Two days later, whole blood was
collected from these mice via heart puncture and the cells were
stained for human CD3 and CD20 and then analyzed by FACS for GFP
expression. In the lower panels, the shaded line illustrates no
virus treatment and the dashed line shows treatment with
FUGW/b12+SINmu virus. The solid line shows treatment with
FUGW/.alpha.CD20+SINmu virus.
[0038] FIG. 7 illustrates expression of a gene of interest using
virus prepared from three different viral constructs. Fresh,
unfractionated human PBMCs (2.times.10.sup.6) were transduced by
co-culturing with concentrated FUGW/.alpha.CD20+SINmu,
CCMV/.alpha.CD20+SINmu or CPGK/.alpha.CD20+SINmu (10.times.10.sup.6
TU). PMA (50 ng/mL) and ionomycin (500 ng/mL) was added into the
culture media to enhance T cells survival and growth. After two
days, the T cell population was identified by co-staining of human
CD3. The solid line shows analysis on transduced cells; the shaded
line illustrates analysis on cells without transduction (as a
control).
DETAILED DESCRIPTION
[0039] Targeting efficient gene delivery vehicles to the desired
cell types with specificity greatly enhances the therapeutic
potential of virus-mediated gene therapy and alleviates concerns of
off-target effects in vivo. In addition, it has many advantages in
other contexts, such as in the generation of transgenic animals
with particular traits, such as disease resistance or production of
a protein in specific tissues.
[0040] The preferred embodiments of the methods and constructs
described herein are based, in part, on the finding that the viral
binding and fusion functions can be separated into two distinct
components (fusogenic proteins and affinity molecules) that are
inserted into the viral envelope. This allows precise targeting of
virus to the desired target cells and efficient transduction and
delivery of a desired polynucleotide or other molecule. An
advantage of this method over others, for example those where viral
fusion proteins are engineered with a foreign binding component, is
that the fusion protein can maintain its biological activity so
that viral titer is not sacrificed for increased target
specificity.
[0041] As discussed in detail below, the methods are preferably
based on the use of recombinant retroviruses, such as lentiviruses,
because these viruses readily incorporate into their envelope
whatever proteins are found on the surface of virus-producing
cells. However, other types of viruses may be used and the methods
modified accordingly. Generally, a packaging cell line is
transfected with one or more vectors encoding the retroviral
components, a gene of interest, a fusion molecule and an affinity
molecule. During budding of the virus the fusion molecule and
affinity molecule, which have been expressed in the packaging cell
membrane, are incorporated into the viral envelope (FIG. 1). As a
result, the retroviral particles comprise a core including the gene
of interest and an envelope comprising the fusion molecule and the
affinity molecule on its surface. The affinity molecule then
recognizes a constituent of the target cell membrane and attaches
the lentivirus to the cell surface (FIG. 2). The binding induces
endocytosis of the target, bringing the lentivirus into an
endosome. There, the fusogenic molecule (FM) triggers membrane
fusion, allowing the virus core to enter the cytosol. Following
reverse transcription and migration of the product to the nucleus,
the genome of the virus integrates into the target cell genome,
incorporating the transgene.
[0042] The methods disclosed herein may be readily adopted to a
variety of affinity molecules and fusogenic molecules. In a
preferred embodiment, the fusion molecule (FM) is preferably a
viral glycoprotein that mediates fusion, preferably in response to
the low pH environment of the endosome, and the affinity molecule
is preferably a membrane-bound protein that is efficiently
endocytosed after binding. The fusion molecule preferably exhibits
fast enough kinetics that the viral contents can empty into the
cytosol before the degradation of the viral particle. In addition,
the fusion molecule can be modified to reduce their binding ability
and thus reduce or eliminate any non-specific binding. That is, by
reducing the binding ability of the fusion molecules, binding of
the virus to the target cell is determined predominantly or
entirely by the affinity molecule, allowing for high target
specificity and reducing undesired effects.
[0043] Affinity molecules may include, for example, antibodies to a
particular antigen on the target cell surface, as well as receptors
for cell surface ligands or ligands for cell surface receptors. The
affinity molecules are preferably membrane bound. Thus, if an
antibody is to be used it may be modified to membrane bound form.
For example, the variable regions from an antibody with the desired
specificity can be cloned into IgG.sub.1. Alternatively, a
transmembrane domain may be attached to an antibody, such as a
single chain antibody.
[0044] In some embodiments, the methods are used to target
dendritic cells using a membrane-bound monoclonal antibody against
the DEC-205 receptor as the affinity molecule. In other
embodiments, incorporation of a membrane-bound form of stem cell
factor as the affinity molecule may be used to target
c-kit-positive cells.
[0045] The modular flexibility (combination of affinity molecule
and fusogenic molecule) and breadth (availability of, for example,
monoclonal antibodies or ligands for many endocytosed cell-specific
surface molecules, and the ability to generate such antibodies) of
the disclosed method is thus especially advantageous in
facilitating the application of targeted gene delivery for therapy,
industry and research. For example, the methods of the present
invention may be used to target tumor cells and deliver a toxic
gene. In another embodiment, cells infected by a pathogen (or
susceptible to such infection) may be targeted to deliver siRNA to
inhibit a stage in the pathogen's life cycle. In still another
embodiment, a cell lacking a particular component (e.g., an enzyme)
may be targeted to deliver a gene encoding for that particular
component.
Definitions
[0046] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g., Singleton et al., Dictionary of Microbiology and Molecular
Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994);
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). Any methods,
devices and materials similar or equivalent to those described
herein can be used in the practice of this invention.
[0047] As used herein, the terms nucleic acid, polynucleotide and
nucleotide are interchangeable and refer to any nucleic acid,
whether composed of phosphodiester linkages or modified linkages
such as phosphotriester, phosphoramidate, siloxane, carbonate,
carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate or sultone linkages,
and combinations of such linkages.
[0048] The terms nucleic acid, polynucleotide and nucleotide also
specifically include nucleic acids composed of bases other than the
five biologically occurring bases (adenine, guanine, thymine,
cytosine and uracil).
[0049] As used herein, a nucleic acid molecule is said to be
"isolated" when the nucleic acid molecule is substantially
separated from contaminant nucleic acid molecules encoding other
polypeptides.
[0050] "Immunization" refers to the provision of antigen to a host.
In some embodiments, antigen is provided to antigen-presenting
cells, such as dendritic cells. For example, as described below,
recombinant virus comprising a gene encoding an antigen can be
targeted to dendritic cells with an affinity molecule specific to a
protein on dendritic cells. Thus the antigen to which an immune
response is desired can be delivered to the dendritic cells. Other
methods of immunization are well known in the art.
[0051] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules that lack antigen specificity. Polypeptides of the latter
kind are, for example, produced at low levels by the lymph system
and at increased levels by myelomas.
[0052] "Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins, composed of two identical light (L)
chains and two identical heavy (H) chains. Each light chain is
linked to a heavy chain by a disulfide bond. The number of
disulfide linkages varies among the heavy chains of different
immunoglobulin isotypes. Each heavy chain comprises a variable
domain (V.sub.H) followed by a number of constant domains. Each
light chain comprises a variable domain at one end (V.sub.L) and a
constant domain at its other end. The constant domain of the light
chain is aligned with the first constant domain of the heavy chain,
and the light chain variable domain is aligned with the variable
domain of the heavy chain.
[0053] The term "antibody" is used in the broadest sense and
specifically covers human, non-human (e.g. murine) and humanized
monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multi-specific antibodies
(e.g., bispecific antibodies), single-chain antibodies, and
antibody fragments so long as they exhibit the desired biological
activity.
[0054] "Target cells" are any cells in which expression of a gene
of interest is desired. Preferably, target cells exhibit a protein
or other molecule on their surface that allows the cell to be
targeted with an affinity molecule, as described below.
[0055] The term "mammal" is defined as an individual belonging to
the class Mammalia and includes, without limitation, humans,
domestic and farm animals, and zoo, sports, and pet animals, such
as sheep, dogs, horses, cats and cows.
[0056] A "subject" or "patient" is any animal, preferably a mammal,
that is in need of treatment.
[0057] As used herein, "treatment" is a clinical intervention made
in response to a disease, disorder or physiological condition
manifested by a patient or to be prevented in a patient. The aim of
treatment includes the alleviation and/or prevention of symptoms,
as well as slowing, stopping or reversing the progression of a
disease, disorder, or condition. "Treatment" refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already affected by a
disease or disorder or undesired physiological condition as well as
those in which the disease or disorder or undesired physiological
condition is to be prevented. "Treatment" need not completely
eliminate a disease, nor need it completely prevent a subject from
catching the disease or disorder.
[0058] "Tumor," as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0059] The term "cancer" refers to a disease or disorder that is
characterized by unregulated cell growth. Examples of cancer
include, but are not limited to, carcinoma, lymphoma, blastoma and
sarcoma. Examples of specific cancers include, but are not limited
to, lung cancer, colon cancer, breast cancer, testicular cancer,
stomach cancer, pancreatic cancer, ovarian cancer, liver cancer,
bladder cancer, colorectal cancer, and prostate cancer. Additional
cancers are well known to those of skill in the art.
[0060] A "vector" is a nucleic acid that is capable of transporting
another nucleic acid. Vectors may be, for example, plasmids,
cosmids or phage. An "expression vector" is a vector that is
capable of directing expression of a protein encoded by one or more
genes carried by the vector when it is present in the appropriate
environment.
[0061] The term "regulatory element" and "expression control
element" are used interchangeably and refer to nucleic acid
molecules that can influence the transcription and/or translation
of an operably linked coding sequence in a particular environment.
These terms are used broadly and cover all elements that promote or
regulate transcription, including promoters, core elements required
for basic interaction of RNA polymerase and transcription factors,
upstream elements, enhancers, and response elements (see, e.g.,
Lewin, "Genes V" (Oxford University Press, Oxford) pages 847-873).
Exemplary regulatory elements in prokaryotes include promoters,
operator sequences and a ribosome binding sites. Regulatory
elements that are used in eukaryotic cells may include, without
limitation, promoters, enhancers, splicing signals and
polyadenylation signals.
[0062] The term "transfection" refers to the introduction of a
nucleic acid into a host cell.
[0063] "Retroviruses" are viruses having an RNA genome.
[0064] "Lentivirus" refers to a genus of retroviruses that are
capable of infecting dividing and non-dividing cells. Several
examples of lentiviruses include HIV (human immunodeficiency virus:
including HIV type 1, and HIV type 2), the etiologic agent of the
human acquired immunodeficiency syndrome (AIDS); visna-maedi, which
causes encephalitis (visna) or pneumonia (maedi) in sheep, the
caprine arthritis-encephalitis virus, which causes immune
deficiency, arthritis, and encephalopathy in goats; equine
infectious anemia virus, which causes autoimmune hemolytic anemia,
and encephalopathy in horses; feline immunodeficiency virus (FIV),
which causes immune deficiency in cats; bovine immune deficiency
virus (BIV), which causes lymphadenopathy, lymphocytosis, and
possibly central nervous system infection in cattle; and simian
immunodeficiency virus (SIV), which cause immune deficiency and
encephalopathy in sub-human primates.
[0065] A "hybrid virus" as used herein refers to a virus having
components from one or more other viral vectors, including element
from non-retroviral vectors, for example, adenoviral-retroviral
hybrids. As used herein hybrid vectors having a retroviral
component are to be considered within the scope of the
retroviruses.
[0066] A lentiviral genome is generally organized into a 5' long
terminal repeat (LTR), the gag gene, the pol gene, the env gene,
the accessory genes (nef, vif, vpr, vpu) and a 3' LTR. The viral
LTR is divided into three regions called U3, R and U5. The U3
region contains the enhancer and promoter elements. The U5 region
contains the polyadenylation signals. The R (repeat) region
separates the U3 and U5 regions and transcribed sequences of the R
region appear at both the 5' and 3' ends of the viral RNA. See, for
example, "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed.,
Oxford University Press, (2000)), O Narayan and Clements J. Gen.
Virology 70:1617-1639 (1989), Fields et al. Fundamental Virology
Raven Press. (1990), Miyoshi H, Blomer U, Takahashi M, Gage F H,
Verma I M. J Virol. 72(10):8150-7 (1998), and U.S. Pat. No.
6,013,516.
[0067] Lentiviral vectors are known in the art, including several
that have been used to transfect hematopoietic stem cells. Such
vectors can be found, for example, in the following publications,
which are incorporated herein by reference: Evans J T et al. Hum
Gene Ther 1999;10:1479-1489; Case S S, Price M A, Jordan C T et al.
Proc Natl Acad Sci USA 1999;96:2988-2993; Uchida N, Sutton R E,
Friera A M et al. Proc Natl Acad Sci USA 1998;95:11939-11944;
Miyoshi H, Smith K A, Mosier D E et al. Science 1999;283:682-686;
Sutton R E, Wu H T, Rigg R et al. Human immunodeficiency virus type
1 vectors efficiently transduce human hematopoietic stem cells. J
Virol 1998;72:5781-5788.
[0068] "Virion," "viral particle" and "retroviral particle" are
used herein to refer to a single virus comprising an RNA genome,
pol gene derived proteins, gag gene derived proteins and a lipid
bilayer displaying an envelope (glyco)protein. The RNA genome is
usually a recombinant RNA genome and thus may contain an RNA
sequence that is exogenous to the native viral genome. The RNA
genome may also comprise a defective endogenous viral sequence.
[0069] A "pseudotyped" retrovirus is a retroviral particle having
an envelope protein that is from a virus other than the virus from
which the RNA genome is derived. The envelope protein may be from a
different retrovirus or from a non-retroviral virus. A preferred
envelope protein is the vesicular stomatitius virus G (VSV G)
protein. However, to eliminate the possibility of human infection,
viruses can alternatively be pseudotyped with an ecotropic envelope
protein that limits infection to a specific species, such as mice
or birds. For example, in one embodiment, a mutant ecotropic
envelope protein is used, such as the ecotropic envelope protein
4.17 (Powell et al. Nature Biotechnology 18(12):1279-1282
(2000)).
[0070] A "self-inactivating 3' LTR" is a 3' long terminal repeat
(LTR) that contains a mutation, substitution or deletion that
prevents the LTR sequences from driving expression of a downstream
gene. A copy of the U3 region from the 3' LTR acts as a template
for the generation of both LTR's in the integrated provirus. Thus,
when the 3' LTR with an inactivating deletion or mutation
integrates as the 5' LTR of the provirus, no transcription from the
5' LTR is possible. This eliminates competition between the viral
enhancer/promoter and any internal enhancer/promoter.
Self-inactivating 3' LTRs are described, for example, in Zufferey
et al. J Virol. 72:9873-9880 (1998), Miyoshi et al. J Virol.
72:8150-8157 and Iwakuma et al. Virology 261:120-132 (1999).
[0071] "Transformation," as defined herein, describes a process by
which exogenous DNA enters a target cell. Transformation may rely
on any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell and may
include, but is not limited to, viral infection, electroporation,
heat shock, lipofection, and particle bombardment. "Transformed"
cells include stably transformed cells in which the inserted DNA is
capable of replication either as an autonomously replicating
plasmid or as part of the host chromosome. Also included are cells
that transiently express a gene of interest.
[0072] A "fusogenic molecule," as described herein, is any molecule
on a viral surface that triggers membrane fusion, and allows the
virus core to pass through the membrane and, typically, enter the
cytosol of a target cell. Viral glycoproteins are one example of
fusogenic molecules.
[0073] An "affinity molecule," or "cell-specific binding
determinant" as described herein, is any molecule on a viral
surface that functions to recognize a molecular constituent on a
target cell membrane and thereby target the virus to the cell
surface. The affinity molecule is most preferably discrete from the
fusogenic molecule.
[0074] By "transgene" is meant any nucleotide sequence,
particularly a DNA sequence, that is integrated into one or more
chromosomes of a host cell by human intervention, such as by the
methods of the present invention. The transgene preferably
comprises a "gene of interest." In other embodiments the transgene
can be a nucleotide sequence, preferably a DNA sequence, that is
used to mark the chromosome where it has integrated. The transgene
does not have to comprise a gene that encodes a protein that can be
expressed.
[0075] A "gene of interest" is not limited in any way and may be
any nucleic acid, without limitation, that is desired to be
integrated, transcribed, translated, and/or expressed in a target
cell. The gene of interest may encode a functional product, such as
a protein or an RNA molecule. Preferably the gene of interest
encodes a protein or other molecule the expression of which is
desired in the host cell. The gene of interest is generally
operatively linked to other sequences that are useful for obtaining
the desired expression of the gene of interest, such as
transcriptional regulatory sequences.
[0076] A "functional relationship" and "operably linked" mean,
without limitation, that the gene is in the correct location and
orientation with respect to the promoter and/or enhancer that
expression of the gene will be affected when the promoter and/or
enhancer is contacted with the appropriate molecules.
[0077] An "RNA coding region" is a nucleic acid that can serve as a
template for the synthesis of an RNA molecule, such as an siRNA.
Preferably, the RNA coding region is a DNA sequence.
[0078] A "small interfering RNA" or "siRNA" is a double-stranded
RNA molecule that is capable of inhibiting the expression of a gene
with which it shares homology. In one embodiment the siRNA may be a
"hairpin" or stem-loop RNA molecule, comprising a sense region, a
loop region and an antisense region complementary to the sense
region. In other embodiments the siRNA comprises two distinct RNA
molecules that are non-covalently associated to form a duplex.
[0079] "2A sequences" or elements are small peptides introduced as
a linker between two proteins, allowing autonomous intraribosomal
self-processing of polyproteins (de Felipe. Genetic Vaccines and
Ther. 2:13 (2004); deFelipe et al. Traffic 5:616-626 (2004)). The
short peptides allow co-expression of multiple proteins from a
single vector, such as co-expression of a fusogenic molecule and
affinity molecule from the same vector. Thus, in some embodiments
polynucleotides encoding the 2A elements are incorporated into a
vector between polynucleotides encoding proteins to be
expressed.
Fusogenic Molecules
[0080] Fusogenic molecules (FMs) are molecules that are able to be
incorporated in the envelope of recombinant viruses and, under the
right conditions, induce membrane fusion allowing entry of a gene
of interest to a target cell. Fusogenic molecules preferably are
able to pseudotype virus, preferably recombinant lentivirus and
thus are able to be incorporated in the viral envelope. Preferably,
the FM does not mediate viral infection of target cells directly,
but still maintains its capability to induce fusion once a virus
enters the endocytic pathways. Thus, while FMs may natively have
the ability to bind a cell surface molecule, FMs with low or
reduced binding ability are preferred to reduce non-specific
transduction. Preferred FMs are viral glycoproteins. In addition,
FMs are preferably resistant to ultracentrifugation to allow
concentration, which is important for in vivo gene delivery.
[0081] FMs preferably induce membrane fusion at a low pH,
independently of affinity molecule binding. Thus, in the disclosed
methods FM induced membrane fusion preferably occurs once the virus
comprising the FM is inside the endosome of a target cell and the
viral core component, including a gene of interest, is delivered to
the cytosol.
[0082] In some embodiments a tag sequence is incorporated into the
fusogenic molecule to allow detection of FM expression and the
presence of the FM in viral particles.
[0083] There are two recognized classes of viral fusogens and both
can be used as FMs (D. S. Dimitrov, Nature Rev. Microbio. 2, 109
(2004)). The class I fusogens trigger membrane fusion using helical
coiled-coil structures whereas the class II fusogens trigger fusion
with .beta. barrels. These two structures have different mechanics
and kinetics (D. S. Dimitrov, Nature Rev. Microbio. 2, 109
(2004)).
[0084] Some non-limiting examples of surface glycoproteins that may
be used as fusion molecules include glycoproteins from
alphaviruses, such as Semliki Forest virus (SFV), Ross River virus
(RRV) and Aura virus (AV), which comprise surface glycoproteins
such as E1, E2, and E3. The E2 glycoproteins derived from the
Sindbis virus (SIN) and the hemagglutinin (HA) of influenza virus
are non-retroviral glycoproteins that recognize particular
molecules on cell surfaces (heparin sulfate glycosaminoglycan for
E2, sialic acid for HA) and are used as FMs in some embodiments.
Their fusion is relatively independent of binding to receptor
molecules, and the activation of fusion is accomplished through
acidification in the endosome (Skehel and Wiley, Annu. Rev.
Biochem. 69, 531-569 (2000); Smit, J. et al. J. Virol. 73,
8476-8484 (1999)). Moreover, they can tolerate certain genetic
modifications and remain efficiently assembled on the retroviral
surface (Morizono et al. J. Virol. 75, 8016-8020). Because of the
ubiquitous presence of surface molecules recognized by some FMs,
binding-defective but fusion competent fusogenic proteins are
preferably used, such as a binding-defective form of HA.
[0085] In other embodiments of the invention, surface glycoproteins
of Lassa fever virus, Hepatitis B virus, Rabies virus, Borna
disease virus, Hantaan virus, or SARS-CoV virus may also be
utilized as fusion molecules. In other embodiments, DV glycoprotein
may be utilized as a fusion molecule.
[0086] In other embodiments of the invention, flavivirus-based
surface glycoproteins may be used as fusion molecules. Like
alphaviruses, flaviviruses use the class II fusion molecule to
mediate infection (Mukhopadhyay et al. (2005) Rev. Microbio. 3,
13-22). prM (about 165 amino acids) and E (about 495 amino acids)
are the glycoproteins of flaviviruses. Also, the ligand-binding
pocket for DV has been well-characterized. Of interest, DC-SIGN
(dendritic-cell-specific ICAM-grabbing non-integrin), a
mannose-specific lectin, has been suggested to specifically
interact with the carbohydrate residues on the DV E protein to
enhance viral entry (Mukhopadhyay et al. (2005) Nat. Rev. Microbio.
3, 13-22). This, lentiviruses enveloped only by DV E protein can
potentially target DCs. The ligand-binding pockets of TBE and DV E
proteins, as well as other fusion molecules described, may be
engineered to be binding deficient and fusion competent in the
following manner.
[0087] In some embodiments, hemagglutinin (HA) from influenza
A/fowl plague virus/Rostock/34 (FPV), a class I fusogen, is used.
(T. Hatziioannou, S. Valsesia-Wittmann, S. J. Russell, F. L.
Cosset, J. Virol. 72, 5313 (1998)). Preferably, a binding defective
version of FPV HA, such as HAmu (FIG. 3A), is used (A. H. Lin et
al., Hum. Gene. Ther. 12, 323 (2001)). HAmu-mediated fusion is
generally considered to be independent of receptor binding (D.
Lavillette, S. J. Russell, F. L. Cosset, Curr. Opin. Biotech. 12,
461 (2001)).
[0088] In other embodiments, a class II FM is used, preferably the
Sindbis virus glycoprotein from the alphavirus family (K. S. Wang,
R. J. Kuhn, E. G. Strauss, S. Ou, J. H. Strauss, J. Virol. 66, 4992
(1992)), herein also referred to as SIN. SIN includes two
transmembrane proteins (S. Mukhopadhyay, R. J. Kuhn, M. G.
Rossmann, Nature Rev. Microbio. 3, 13 (2005)), a first protein
responsible for fusion (E1), and a second protein for cell binding
(E2). SIN is known to pseudotype both oncoretroviruses and
lentiviruses.
[0089] In some embodiments a binding-deficient and fusion-competent
SIN is used as the fusogenic molecule. For example, a SIN fusogenic
molecule can be used in which the immunoglobulin G binding domain
of protein A (ZZ domain) is incorporated into the E2 protein and
one or more additional mutations are made to inactivate the
receptor binding sites (K. Morizono et al., Nature Med. 11, 346
(2005)).
[0090] The gene encoding the FM is preferably cloned into an
expression vector, such as pcDNA3 (Invitrogen). Packaging cells,
such as 293T cells are then co-transfected with the viral vector
comprising the gene of interest, a packaging vector (if necessary),
one or more vectors encoding an affinity molecule and any
associated components, and the vector for expression of the
fusogenic molecule. The FM is expressed on the membrane of the
packaging cell and incorporated into the recombinant virus.
Expression of envelope glycoprotein on the packaging cell surface
may be analyzed by FACS.
[0091] Based on information obtained, for example from structural
studies and molecular modeling, mutagenesis may be employed to
generate the mutant forms of glycoproteins that maintain their
fusogenic ability but have the desired level of binding. Several
mutants may be created for each glycoprotein and assayed using the
methods described below, or other methods known in the art, to
identify FMs with the most desirable characteristics.
[0092] To select suitable FMs (either wild-type or mutant), viruses
bearing both the FM and an affinity molecule are prepared and
tested for their selectivity and/or their ability to facilitate
penetration of the target cell membrane. Viruses that do not
display the affinity molecule may be used as controls for measuring
selectivity, while viruses displaying the affinity molecule and
wild-type glycoprotein can be used as controls for examining titer
effects in mutants. Cells expressing the binding partner of the
affinity molecule are transduced by the virus using a standard
infection assay. After a specified time, for example, 48 hours
post-transduction, cells can be collected and the percentage of
cells infected by the virus comprising the mutant FM can be
determined by, for example, FACS. The selectivity can be scored by
calculating the percentage of cells infected by virus. Similarly,
the effect of mutations on viral titer can be quantified by
dividing the percentage of cells infected by virus comprising a
mutant FM by the percentage of cells infected by virus comprising
the corresponding wild type FM. A preferred mutant will give the
best combination of selectivity and infectious titer. Once an FM is
selected, viral concentration assays may be performed to confirm
that viruses enveloped by the FM can be concentrated. Viral
supernatants are collected and concentrated by ultracentrifugation.
The titers of viruses can be determined by limited dilution of
viral stock solution and transduction of cells expressing the
binding partner of the affinity molecule.
[0093] In some embodiments, BlaM-Vpr fusion protein may be utilized
to evaluate viral penetration, and thus the efficacy of a fusion
molecule (wild-type or mutant). An affinity molecule, such as an
antibody, may envelope viral particles incorporating BlaM-Vpr. Such
virus may be prepared, for example, by transient transfection of
packaging cells with one or more vectors comprising the viral
elements, BlaM-Vpr, the FM of interest and an affinity molecule.
The resulting viruses can be used to infect cells expressing a
molecule recognized by the affinity molecule in the absence or
presence of the free inhibitor of affinity molecule binding (such
as an antibody). Cells can then be washed with CO.sub.2-independent
medium and loaded with CCF2 dye (Aurora Bioscience). After
incubation at room temperature to allow completion of the cleavage
reaction, the cells can be fixed by paraformaldehyde and analyzed
by FACS and microscopy. The presence of blue cells indicates the
penetration of viruses into the cytoplasm; fewer blue cells would
be expected when blocking antibody is added.
[0094] To investigate whether penetration is dependent upon a low
pH, and select FM with the desired pH dependence, NH.sub.4Cl or
other compound that alters pH can be added at the infection step
(NH.sub.4Cl will neutralize the acidic compartments of endosomes).
In the case of NH.sub.4Cl, he disappearance of blue cells will
indicate that penetration of viruses is low pH-dependent.
[0095] In addition, to confirm that the fusion molecule activity is
pH-dependent, lysosomotropic agents, such as ammonium chloride,
chloroquine, concanamycin, bafilomycin A.sub.1, monensin,
nigericin, etc., may be added into the incubation buffer. These
agents can elevate the pH within the endosomal compartments (e.g.,
Drose and Altendorf, J. Exp. Biol. 200, 1-8, 1997). The inhibitory
effect of these agents will reveal the role of pH for viral fusion
and entry. The different entry kinetics between viruses displaying
different fusogenic molecules may be compared and the most suitable
selected for a particular application.
[0096] PCR entry assays may be utilized to monitor reverse
transcription and thus measure kinetics of viral DNA synthesis as
an indication of the kinetics of viral entry. For example, viral
particles comprising a particular FM and an affinity molecule may
be incubated with packaging cells, such as 293T cells, expressing
the appropriate cognate for the affinity molecule. Either
immediately, or after incubation (to allow infection to occur)
unbound viruses are removed and aliquots of the cells are analyzed.
DNA may then be extracted from these aliquots and semi-quantitative
performed using LTR-specific primers. The appearance of
LTR-specific DNA products will indicate the success of viral entry
and uncoating.
Cell-Specific Binding Determinants (Membrane-Bound Affinity
Molecules)
[0097] In preferred embodiments of the invention, the viral surface
comprises a cell-specific binding determinant comprising a
membrane-bound affinity molecule. The affinity molecule is selected
to bind selectively to a surface molecule, for example, a receptor
protein, on a target cell. Preferably, the binding is high-affinity
binding and in some embodiments the dissociation constant may be in
the range of 10.sup.-6 to 10.sup.-12 M. However, in other
embodiments lower or higher affinity binding is possible.
[0098] Generally, the target cell protein is one that is expressed
selectively on the target cells. That is, the target cell protein
is preferably expressed exclusively on target cells or expressed on
target cells at a higher concentration than on other cells.
However, in some embodiments where a wide population of target
cells is to be targeted, a target cell protein may be expressed in
a variety of cell types, or even ubiquitously expressed.
[0099] The affinity molecule is preferably a membrane-bound ligand,
membrane bound receptor, or a membrane bound antibody, such as,
without limitation, a chimeric antibody, an antibody fragment or a
single chain antibody. Although referred to generally in the
singular, the affinity molecule may comprise two or more molecules,
such as an IgG.sub.1 and the Ig.alpha. and Ig.beta. molecules.
[0100] The membrane-bound affinity molecule is most preferably a
separate molecule from the fusogenic molecule.
[0101] Upon binding of the affinity molecule to a molecule on the
target cell surface, the virus particle is taken up by endocytosis.
In some embodiments, a pH change then induces the fusion molecule
to fuse and allows the delivery of the viral core across the
membrane and into the cytosol.
[0102] The affinity molecule is co-expressed in the packaging cell
with the virus comprising the gene of interest and a fusogenic
molecule. Constructs for expressing the affinity molecule in the
packaging cell also may include, in some embodiments, sequences
encoding necessary signal peptides to direct the protein to be
expressed on the cell surface.
[0103] Packaging cells, such as 293T cells, are preferably
co-transfected with the viral vector comprising the gene of
interest, a packaging vector (if necessary), one or more vectors
encoding the affinity molecule and any associated components (such
as Ig.alpha. and Ig.beta. ), and the vector for expression of the
fusogenic molecule. In some embodiments, two or more of these
components are combined in a multi-cistronic expression vector. For
example, in some embodiments the fusogenic molecules and the
affinity molecule are included in the same vector.
[0104] The affinity molecule is expressed on the membrane of the
packaging cell and incorporated into the envelope of the
recombinant virus. Expression of the affinity molecule may be
assayed by known methods, such as with an antibody to the affinity
molecule.
[0105] The ability of various types of affinity molecules (such as
antibodies to different epitopes of the target protein) to
facilitate targeting of the recombinant retrovirus and gene
delivery can be compared and the molecule with the most desirable
characteristics can be selected. Desirable characteristics include,
for example, the ability to stimulate efficient endocytosis of the
virus, the ability to bind the target molecule with specificity
and, similarly, the ability to facilitate binding of the
recombinant virus to the target cell. For example, the same target
bound by different affinity molecules can be endocytosed
differently. Thus, in some embodiments, an affinity molecule with
the ability to trigger endocytosis while maintaining binding
specificity for a particular target cell type is selected.
Similarly, the same antibody-antigen pair could stimulate different
endocytosis pathways in different cells, leading to a different
ability to facilitate delivery of the gene of interest in the
different cells. The assays discussed above with respect to the
fusogenic molecule can be modified to compare the abilities of
various antibodies, or other affinity molecules, to mediate
delivery of a gene of interest to a particular target cell.
[0106] The transduction efficiency mediated by different affinity
molecules, for example, different anti-CD20 antibodies in different
cells, can readily be tested. Transduction can be performed in
cells most closely resembling the target cells (or the target cells
if available) and efficiency of viral entry quantified and
compared. The above BalM-Vpr-based assay and PCR-based assay may be
employed to measure the efficiency of viral entry for the different
affinity molecules in different cell lines. Furthermore, confocal
microscopy can be utilized to investigate the endocytosis induced
by different affinity molecules and in different cell types. These
detailed studies can assist in determining correlation between
transduction efficiency and endocytosis behavior, which can be used
to optimize targeting schemes.
Antibodies as Affinity Molecules
[0107] In some embodiments the cell-specific binding determinant is
an antibody. Antibodies may be generated against any desired cell
surface molecule on target cells using well known methods.
Antibodies may be generated in an appropriate species to minimize
immune response. In other embodiments humanized or chimeric
antibodies are prepared and used. For example, antibodies may be
generated in a mouse, rabbit or other animal and the variable
regions combined with the constant regions of a human antibody. In
addition, many antibodies are commercially available and can be
selected based on the target molecule on the target cells.
[0108] The cell-specific binding determinant is preferably membrane
bound or otherwise associated with the membrane of the packaging
cell and ultimately the envelope of the recombinant virus. Thus,
when the affinity molecule is a soluble antibody it may be
necessary to modify the antibody to cause it to associate with the
membrane.
[0109] In some preferred embodiments, an antibody used as an
affinity molecule preferably includes variable regions from an
antibody to the target and constant regions from human IgG.sub.1
That is, the variable regions of an antibody to the target, such as
a soluble antibody are combined with the constant regions of
IgG.sub.1 to make a membrane bound chimeric immunoglobulin with the
desired specificity. If the recombinant retrovirus is to be used in
humans, the IgG.sub.1 is preferably human. Methods for preparing
chimeric antibodies are well known in the art.
[0110] In such embodiments the packaging cell (used to assemble the
virus) is preferably cotransfected with vectors encoding both the
heavy and light chains of IgG.sub.1 along with vectors for the
expression of immunoglobulin alpha (Ig.alpha.) and immunoglobulin
beta (Ig.beta.). Immunoglobulin alpha (Ig.alpha.) and
immunoglobulin beta (Ig.beta.) help to bind the antibody to the
surface membrane of the virus. In some embodiments the antibody is
generated in a species other than human, such as mouse or rabbit,
and the variable regions are cloned into human IgG.sub.1.
[0111] In some embodiments, a multicistronic vector is used to
direct expression of an antibody and Ig.alpha. and Ig.beta..
Assembly PCR may be employed to link the antibody heavy chain,
light chain, and Ig.alpha. and Ig.beta.. In one embodiment the
genes encoding the three components are separated by three 2A
peptides, to facilitate expression from a single promoter. Then,
the entire cassette can be cloned into an expression vector such as
pCDNA3 (Invitrogen). The efficiency of antibody expression on the
surface of packaging cells can be analyzed by FACS staining.
[0112] In other embodiments, the affinity molecule is a single
chain antibody, either natural or chimeric, and is preferably fused
with a transmembrane domain from another protein (Chou et. al.,
Biotechnol. Bioeng., 65, 160 (1999); Liao et al, Biotechnol.
Bioeng., 73, 313 (2001); de Ines et al, J. Immunol., 163, 3948
(1999); Lee et al, J. Immunol., 173, 4618 (2004)). A single chain
membrane-bound form of antibody (scAbm) is advantageous in
simplifying viral production because display of the natural form of
an antibody on the cell surface requires co-expression of four
genes: antibody heavy chain, antibody light chain, Ig.alpha. and
Ig.beta..
[0113] scAbms are typically designed to have heavy chain and light
chain variable domains linked by a flexible peptide linker. They
also carry a signal peptide at their N terminus and a transmembrane
domain at their C terminus for anchoring to the cell surface. In
some embodiments a slightly different version of scAbm is used,
comprising heavy chain and light chain variable domains of the
desired antibody (specific to an antigen on the target cell
surface) linked by a peptide such as the (GGGGSGGGS).sub.2 peptide,
and a dimerization region including the hinge CH2-CH3 domain of
human IgG1, and the transmembrane domain and the cytoplasmic tail
of the human HLA-A2 to display this chimeric protein on the cell
surface.
[0114] Thus, in some embodiments a target molecule is identified
that is expressed, preferably exclusively or predominantly, on a
desired target cell or cell population. Antibodies to the target
molecule are generated using standard methods and the variable
regions are combined with the constant regions of human IgG.sub.1
to form an affinity molecule to direct the recombinant retrovirus
to the target cells with specificity.
Non-Antibody Affinity Molecules
[0115] In other embodiments, the affinity molecule can be a ligand,
preferably a peptide or protein ligand, that binds to a cell
surface receptor on a target cell, a receptor that binds to a
cell-surface ligand on a target cell. When the affinity molecule is
a soluble ligand or receptor, the affinity molecule is preferably
constructed as a fusion with a transmembrane domain or other
component to anchor it to the membrane. Suitable ligands include,
for example, hormones, growth factors, and the like. One specific
example of a non-antibody affinity molecule is stem cell
factor.
[0116] While the affinity molecule can be a naturally occurring
molecule, it can also be a non-natural protein, for example, one
specifically created to bind to a particular protein on a target
cell. In addition, the affinity molecule is not limited to
proteins, but could comprise any compound that is able to
specifically interact with a molecule on the target cell in such a
way as to allow endocytosis of the viral particle into the target
cell. For example, in some embodiments the affinity molecule may
comprise a carbohydrate.
Delivery Vectors
[0117] In a preferred embodiment, one or more vectors are used to
introduce the desired polynucleotides into the target cell. The
vectors comprise the polynucleotide sequences encoding the various
components of the recombinant retrovirus itself, the gene(s) of
interest, the fusogenic molecule and affinity molecule, and any
components necessary for the production of the virus that are not
provided by the packaging cell. These polynucleotides are typically
under the control of one or more regulatory elements that direct
the expression of the coding sequences in the packaging cell and
the target cell, as appropriate. Eukaryotic cell expression vectors
are well known in the art and are available from a number of
commercial sources.
[0118] In some embodiments, packaging cells are transfected with a
viral vector (as discussed below) along with two or more additional
vectors. For example, in addition to the viral vector a second
vector preferably carries a gene encoding a fusogenic molecule,
such as HAmu or SINmu, as described elsewhere in the application. A
third vector preferably carries a gene encoding an affinity
molecule, such as an antibody, as described elsewhere in the
application. Furthermore, in some embodiments, one or more
additional vectors preferably include genes encoding packaging cell
requirements, for example, viral envelope proteins such as pol,
env, and gag. Also, in some embodiments, such as where the affinity
molecule is an IgG.sub.1 immunoglobulin, one or more further
vectors are used that encode accessory proteins, such as genes
encoding Ig.alpha. and Ig.beta..
[0119] In other embodiments, one or more multicistronic expression
vectors are utilized that include two or more of the elements
(e.g., the viral genes, gene(s) of interest, the FM, affinity
molecule, Ig.alpha., Ig.beta.) necessary for production of the
desired recombinant retrovirus in packaging cells. The use of
multicistronic vectors reduces the total number of vectors required
and thus avoids the possible difficulties associated with
coordinating expression from multiple vectors. In a multicistronic
vector the various elements to be expressed are operably linked to
one or more promoters (and other expression control elements as
necessary). For example, in one embodiment a multicistronic
expression vector is used to express an antibody affinity molecule
and associated components. The vector preferably comprises
polynucleotides encoding the antibody heavy and light chain and
Ig.alpha. and Ig.beta.. In other embodiments a multicistronic
vector comprising a gene of interest, a reporter gene, and viral
elements is used. Such a vector may be cotransfected, for example,
along with a multicistronic vector encoding both the FM and
affinity molecules.
[0120] Each component to be expressed in a multicistronic
expression vector may be separated by a an IRES element or a viral
2A element to allow for separate expression of the various proteins
from the same promoter. IRES elements and 2A elements are known in
the art (U.S. Pat. No. 4,937,190; de Felipe et al. Traffic
5:616-626 (2004)). In one embodiment, oligonucleotides encoding
furin cleavage site sequences (RAKR) (Fang et al. Nat. Biotech 23,
584-590 (2005)) linked with 2A-like sequences from foot-and-mouth
diseases virus (FMDV), equine rhinitis A virus (ERAV), and thosea
asigna virus (TaV) (Szymczak et al. (2004) Nat. Biotechnol. 22,
589-594) are used to separate genetic elements in a multicistronic
vector. The efficacy of a particular multicistronic vector for use
in synthesizing the desired recombinant retrovirus can readily be
tested by detecting expression of each of the genes using standard
protocols.
[0121] Generation of the vector(s) can be accomplished using any
suitable genetic engineering techniques known in the art,
including, without limitation, the standard techniques of
restriction endonuclease digestion, ligation, transformation,
plasmid purification, and DNA sequencing, for example as described
in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor Laboratory Press, N.Y. (1989)), Coffin et al.
(Retroviruses. Cold Spring Harbor Laboratory Press, N.Y. (1997))
and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford
University Press, (2000)).
[0122] The vector(s) may incorporate sequences from the genome of
any known organism. The sequences may be incorporated in their
native form or may be modified in any way. For example, the
sequences may comprise insertions, deletions or substitutions.
[0123] Expression control elements that may be used for regulating
the expression of the components are known in the art and include,
but are not limited to, inducible promoters, constitutive
promoters, secretion signals, enhancers and other regulatory
elements.
[0124] In one embodiment, a vector will include a prokaryotic
replicon, i.e., a DNA sequence having the ability to direct
autonomous replication and maintenance of the recombinant DNA
molecule extrachromosomally in a prokaryotic host cell, such as a
bacterial host cell, transformed therewith. Such replicons are well
known in the art. In addition, vectors that include a prokaryotic
replicon may also include a gene whose expression confers a
detectable marker such as a drug resistance. Typical bacterial drug
resistance genes are those that confer resistance to ampicillin or
tetracycline.
[0125] The vector(s) may include one or more genes for selectable
markers that are effective in a eukaryotic cell, such as a gene for
a drug resistance selection marker. This gene encodes a factor
necessary for the survival or growth of transformed host cells
grown in a selective culture medium. Host cells not transformed
with the vector containing the selection gene will not survive in
the culture medium. Typical selection genes encode proteins that
confer resistance to antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, complement auxotrophic
deficiencies, or supply critical nutrients withheld from the media.
The selectable marker can optionally be present on a separate
plasmid and introduced by co-transfection.
[0126] Vectors will usually contain a promoter that is recognized
by the packaging cell and that is operably linked to the
polynucleotide(s) encoding the FM, affinity molecule, viral
components, and the like. A promoter is an expression control
element formed by a DNA sequence that permits binding of RNA
polymerase and transcription to occur. Promoters are untranslated
sequences that are located upstream (5') to the start codon of a
structural gene (generally within about 100 to 1000 bp) and control
the transcription and translation of the antigen-specific
polynucleotide sequence to which they are operably linked.
Promoters may be inducible or constitutive. Inducible promoters
initiate increased levels of transcription from DNA under their
control in response to some change in culture conditions, such as a
change in temperature.
[0127] One of skill in the art will be able to select an
appropriate promoter based on the specific circumstances. Many
different promoters are well known in the art, as are methods for
operably linking the promoter to the gene to be expressed. Both
native promoter sequences and many heterologous promoters may be
used to direct expression in the packaging cell and target cell.
However, heterologous promoters are preferred, as they generally
permit greater transcription and higher yields of the desired
protein as compared to the native promoter.
[0128] The promoter may be obtained, for example, from the genomes
of viruses such as polyoma virus, fowlpox virus, adenovirus, bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). The
promoter may also be, for example, a heterologous mammalian
promoter, e.g., the actin promoter or an immunoglobulin promoter, a
heat-shock promoter, or the promoter normally associated with the
native sequence, provided such promoters are compatible with the
target cell. In one embodiment, the promoter is the naturally
occurring viral promoter in a viral expression system.
[0129] Transcription may be increased by inserting an enhancer
sequence into the vector(s). Enhancers are typically cis-acting
elements of DNA, usually about 10 to 300 bp in length, that act on
a promoter to increase its transcription. Many enhancer sequences
are now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Preferably an enhancer from a
eukaryotic cell virus will be used. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. The enhancer may be spliced into the vector at a
position 5' or 3' to the antigen-specific polynucleotide sequence,
but is preferably located at a site 5' from the promoter.
[0130] Expression vectors will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA.
These sequences are often found in the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs and are
well known in the art.
[0131] Plasmid vectors containing one or more of the components
described above are readily constructed using standard techniques
well known in the art.
[0132] For analysis to confirm correct sequences in plasmids
constructed, the plasmid may be replicated in E. coli, purified,
and analyzed by restriction endonuclease digestion, and/or
sequenced by conventional methods.
[0133] Vectors that provide for transient expression in mammalian
cells may also be used. Transient expression involves the use of an
expression vector that is able to replicate efficiently in a host
cell, such that the host cell accumulates many copies of the
expression vector and, in turn, synthesizes high levels of a the
polypeptide encoded by the antigen-specific polynucleotide in the
expression vector. Sambrook et al., supra, pp. 16.17-16.22.
[0134] Other vectors and methods suitable for adaptation to the
expression of the viral polypeptides are well known in the art and
are readily adapted to the specific circumstances.
[0135] Using the teachings provided herein, one of skill in the art
will recognize that the efficacy of a particular expression system
can be tested by transforming packaging cells with a vector
comprising a gene encoding a reporter protein and measuring the
expression using a suitable technique, for example, measuring
fluorescence from a green fluorescent protein conjugate. Suitable
reporter genes are well known in the art.
[0136] Transformation of packaging cells with vectors of the
present invention is accomplished by well-known methods, and the
method to be used is not limited in any way. A number of non-viral
delivery systems are known in the art, including for example,
electroporation, lipid-based delivery systems including liposomes,
delivery of "naked" DNA, and delivery using polycyclodextrin
compounds, such as those described in Schatzlein AG. 2001.
Non-Viral Vectors in Cancer Gene Therapy: Principles and
Progresses. Anticancer Drugs. Cationic lipid or salt treatment
methods are typically employed, see, for example, Graham et al.
Virol. 52:456, (1973); Wigler et al. Proc. Natl. Acad. Sci. USA
76:1373-76, (1979). The calcium phosphate precipitation method is
preferred. However, other methods for introducing the vector into
cells may also be used, including nuclear microinjection and
bacterial protoplast fusion.
Viral Vector and Packaging Cells
[0137] One of the vectors encodes the core virus (the "viral
vector"). There are a large number of available viral vectors that
are suitable for use with the invention, including those identified
for human gene therapy applications, such as those described in
Pfeifer A, Verma I M. 2001. Gene Therapy: promises and problems.
Annu. Rev. Genomics Hum. Genet. 2:177-211. Suitable viral vectors
include vectors based on RNA viruses, such as retrovirus-derived
vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors,
and include more complex retrovirus-derived vectors, e.g.,
lentivirus-derived vectors. Human Immunodeficiency virus
(HIV-1)-derived vectors belong to this category. Other examples
include lentivirus vectors derived from HIV-2, feline
immunodeficiency virus (FIV), equine infectious anemia virus,
simian immunodeficiency virus (SIV) and maedi/visna virus.
[0138] The viral vector preferably comprises one or more genes
encoding components of the recombinant virus as well as one or more
genes of interest. The viral vector may also comprise genetic
elements that facilitate expression of the gene of interest in a
target cell, such as promoter and enhancer sequences. In order to
prevent replication in the target cell, endogenous viral genes
required for replication may be removed and provided separately in
the packaging cell line.
[0139] In a preferred embodiment the viral vector comprises an
intact retroviral 5' LTR and a self-inactivating 3' LTR.
[0140] Any method known in the art may be used to produce
infectious retroviral particles whose genome comprises an RNA copy
of the viral vector. To this end, the viral vector (along with
other vectors encoding the FM, affinity molecule, etc.) is
preferably introduced into a packaging cell line that packages
viral genomic RNA based on the viral vector into viral
particles.
[0141] The packaging cell line provides the viral proteins that are
required in trans for the packaging of the viral genomic RNA into
viral particles. The packaging cell line may be any cell line that
is capable of expressing retroviral proteins. Preferred packaging
cell lines include 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC
CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL
1430). The packaging cell line may stably express the necessary
viral proteins. Such a packaging cell line is described, for
example, in U.S. Pat. No. 6,218,181. Alternatively a packaging cell
line may be transiently transfected with plasmids comprising
nucleic acid that encodes one or more necessary viral proteins
(along with the viral vector and the vectors encoding the FM and
affinity molecules).
[0142] Viral particles comprising a polynucleotide with the gene of
interest and an envelope comprising the FM and affinity molecules
are collected and allowed to infect the target cell. Target cell
specificity may be further improved by pseudotyping the virus.
Methods for pseudotyping are well known in the art.
[0143] In one embodiment, the recombinant retrovirus used to
deliver the gene of interest is a modified lentivirus and the viral
vector is based on a lentivirus. As lentiviruses are able to infect
both dividing and non-dividing cells, in this embodiment it is not
necessary for target cells to be dividing (or to stimulate the
target cells to divide).
[0144] In another embodiment the vector is based on the murine stem
cell virus (MSCV). The MSCV vector provides long-term stable
expression in target cells, particularly hematopoietic precursor
cells and their differentiated progeny.
[0145] In another embodiment, the vector is based on a modified
Moloney virus, for example a Moloney Murine Leukemia Virus. In a
further embodiment, the vector is based on a Murine Stem Cell Virus
(Hawley, R. G., et al. (1996) Proc. Natl. Acad. Sci. USA
93:10297-10302; Keller, G., et al. (1998) Blood 92:877-887; Hawley,
R. G., et al. (1994) Gene Ther. 1:136-138). The viral vector can
also can be based on a hybrid virus such as that described in Choi,
J K; Hoanga, N; Vilardi, A M; Conrad, P; Emerson, S G; Gewirtz, A
M. (2001) Stem Cells 19, No. 3, 236-246.
[0146] A DNA viral vector may be used, including, for example
adenovirus-based vectors and adeno-associated virus (AAV)-based
vectors. Likewise, retroviral-adenoviral vectors also can be used
with the methods of the invention.
[0147] Other vectors also can be used for polynucleotide delivery
including vectors derived from herpes simplex viruses (HSVs),
including amplicon vectors, replication-defective HSV and
attenuated HSV (Krisky D M, Marconi P C, Oligino T J, Rouse R J,
Fink D J, et al. 1998. Development of herpes simplex virus
replication-defective multigene vectors for combination gene
therapy applications. Gene Ther. 5: 1517-30).
[0148] Other vectors that have recently been developed for gene
therapy uses can also be used with the methods of the invention.
Such vectors include those derived from baculoviruses and
alpha-viruses. (Jolly D J. 1999. Emerging viral vectors. pp 209-40
in Friedmann T, ed. 1999. The development of human gene therapy.
New York: Cold Spring Harbor Lab).
[0149] In the preferred embodiments the viral construct comprises
sequences from a lentivirus genome, such as the HIV genome or the
SIV genome. The viral construct preferably comprises sequences from
the 5' and 3' LTRs of a lentivirus. More preferably the viral
construct comprises the R and U5 sequences from the 5' LTR of a
lentivirus and an inactivated or self-inactivating 3' LTR from a
lentivirus. The LTR sequences may be LTR sequences from any
lentivirus from any species. For example, they may be LTR sequences
from HIV, SIV, FIV or BIV. Preferably the LTR sequences are HIV LTR
sequences.
[0150] The viral construct preferably comprises an inactivated or
self-inactivating 3' LTR. The 3' LTR may be made self-inactivating
by any method known in the art. In the preferred embodiment the U3
element of the 3' LTR contains a deletion of its enhancer sequence,
preferably the TATA box, Spl and NF-kappa B sites. As a result of
the self-inactivating 3' LTR, the provirus that is integrated into
the host cell genome will comprise an inactivated 5' LTR.
[0151] Optionally, the U3 sequence from the lentiviral 5' LTR may
be replaced with a promoter sequence in the viral construct. This
may increase the titer of virus recovered from the packaging cell
line. An enhancer sequence may also be included. Any
enhancer/promoter combination that increases expression of the
viral RNA genome in the packaging cell line may be used. In a
preferred embodiment the CMV enhancer/promoter sequence is
used.
[0152] The viral construct generally comprises a gene that encodes
a protein (or other molecule, such as siRNA) that is desirably
expressed in one or more target cells. Preferably the gene of
interest is located between the 5' LTR and 3' LTR sequences.
Further, the gene of interest is preferably in a functional
relationship with other genetic elements, for example transcription
regulatory sequences such as promoters and/or enhancers, to
regulate expression of the gene of interest in a particular manner
once the gene is incorporated into the target cell. In certain
embodiments, the useful transcriptional regulatory sequences are
those that are highly regulated with respect to activity, both
temporally and spatially.
[0153] Preferably the gene of interest is in a functional
relationship with internal promoter/enhancer regulatory sequences.
An "internal" promoter/enhancer is one that is located between the
5' LTR and the 3' LTR sequences in the viral construct and is
operably linked to the gene that is desirably expressed.
[0154] The internal promoter/enhancer may be any promoter, enhancer
or promoter/enhancer combination known to increase expression of a
gene with which it is in a functional relationship. A "functional
relationship" and "operably linked" mean, without limitation, that
the gene is in the correct location and orientation with respect to
the promoter and/or enhancer that expression of the gene will be
affected when the promoter and/or enhancer is contacted with the
appropriate molecules.
[0155] The internal promoter/enhancer is preferably selected based
on the desired expression pattern of the gene of interest and the
specific properties of known promoters/enhancers. Thus, the
internal promoter may be a constitutive promoter. Non-limiting
examples of constitutive promoters that may be used include the
promoter for ubiquitin, CMV (Karasuyama et al J. Exp. Med. 169:13
(1989), beta-actin (Gunning et al. Proc. Natl. Acad. Sci. USA
84:4831-4835 (1987) and pgk (see, for example, Adra et al. Gene
60:65-74 (1987), Singer-Sam et al. Gene 32:409-417 (1984) and
Dobson et al. Nucleic Acids Res. 10:2635-2637 (1982)).
[0156] Alternatively, the promoter may be a tissue specific
promoter. Several non-limiting examples of tissue specific
promoters that may be used include lck (see, for example, Garvin et
al. Mol. Cell Biol. 8:3058-3064 (1988) and Takadera et al. Mol.
Cell Biol. 9:2173-2180 (1989)), myogenin (Yee et al. Genes and
Development 7:1277-1289 (1993), and thy1 (Gundersen et al. Gene
113:207-214 (1992). In addition, promoters may be selected to allow
for inducible expression of the gene. A number of systems for
inducible expression are known in the art, including the
tetracycline responsive system and the lac operator-repressor
system. It is also contemplated that a combination of promoters may
be used to obtain the desired expression of the gene of interest.
The skilled artisan will be able to select a promoter based on the
desired expression pattern of the gene in the organism and/or the
target cell of interest.
[0157] In some embodiments the viral construct preferably comprises
at least one RNA Polymerase II or III promoters. The RNA Polymerase
II or III promoter is operably linked to the gene of interest and
can also be linked to a termination sequence. In addition, more
than one RNA Polymerase II or III promoters may be
incorporated.
[0158] RNA polymerase II and III promoters are well known to one of
skill in the art. A suitable range of RNA polymerase III promoters
can be found, for example, in Paule and White. Nucleic Acids
Research., Vol 28, pp 1283-1298 (2000), which is hereby
incorporated by reference in its entirety. The definition of RNA
polymerase II or III promoters, respectively, also include any
synthetic or engineered DNA fragment that can direct RNA polymerase
II or III, respectively, to transcribe its downstream RNA coding
sequences. Further, the RNA polymerase II or III (Pol II or III)
promoter or promoters used as part of the viral vector can be
inducible. Any suitable inducible Pol II or III promoter can be
used with the methods of the invention. Particularly suited Pol II
or III promoters include the tetracycline responsive promoters
provided in Ohkawa and Taira Human Gene Therapy, Vol. 11, pp
577-585 (2000) and in Meissner et al. Nucleic Acids Research, Vol.
29, pp 1672-1682 (2001), which are incorporated herein by
reference.
[0159] An internal enhancer may also be present in the viral
construct to increase expression of the gene of interest. For
example the CMV enhancer (Karasuyama et al J. Exp. Med. 169:13
(1989) may be used in combination with the chicken .beta.-actin
promoter. Again, one of skill in the art will be able to select the
appropriate enhancer based on the desired expression pattern.
[0160] The gene of interest is not limited in any way and includes
any nucleic acid that the skilled practitioner desires to have
integrated, transcribed, translated, and/or expressed in the target
cell. For example, the gene of interest may encode a polypeptide,
such as a hormone, toxin or antigen, or encode a nucleotide such as
an siRNA.
[0161] In some embodiments, a gene of interest is incorporated as a
safety measure and allows for the selective killing of infected
target cells within a heterogeneous population, for example within
an animal, such as within a human patient. In one such embodiment,
the gene of interest is a thymidine kinase gene (TK), the
expression of which renders a target cell susceptible to the action
of the drug gancyclovir.
[0162] In addition, more than one gene of interest may be placed in
functional relationship with the internal promoter. For example a
gene encoding a marker protein may be placed after the primary gene
of interest to allow for identification of cells that are
expressing the desired protein. In one embodiment a fluorescent
marker protein, preferably green fluorescent protein (GFP), is
incorporated into the construct along with the gene of interest. If
one or more additional reporter genes is included, internal
ribosomal entry site (IRES) sequences, or 2A elements are also
preferably included, separating the primary gene of interest from a
reporter gene and/or any other gene of interest. The IRES or 2A
sequences may facilitate the expression of the reporter gene, or
other genes.
[0163] The viral construct may also contain additional genetic
elements. The types of elements that may be included in the
construct are not limited in any way and will be chosen by the
skilled practitioner to achieve a particular result. For example, a
signal that facilitates nuclear entry of the viral genome in the
target cell may be included. An example of such a signal is the
HIV-1 flap signal.
[0164] Further, elements may be included that facilitate the
characterization of the provirus integration site in the target
cell. For example, a tRNA amber suppressor sequence may be included
in the construct.
[0165] In addition, the construct may contain one or more genetic
elements designed to enhance expression of the gene of interest.
For example, a woodchuck hepatitis virus responsive element (WRE)
may be placed into the construct (Zufferey et al. J. Virol.
74:3668-3681 (1999); Deglon et al. Hum. Gene Ther. 11: 179-190
(2000)).
[0166] A chicken .beta.-globin insulator may also be included in
the viral construct. This element has been shown to reduce the
chance of silencing the integrated provirus in the target cell due
to methylation and heterochromatinization effects. In addition, the
insulator may shield the internal enhancer, promoter and exogenous
gene from positive or negative positional effects from surrounding
DNA at the integration site on the chromosome.
[0167] Any additional genetic elements are preferably inserted 3'
of the gene of interest.
[0168] In a specific embodiment, the viral vector comprises: a
cytomegalovirus (CMV) enhancer/promoter sequence; the R and U5
sequences from the HIV 5' LTR; the HIV-1 flap signal; an internal
enhancer; an internal promoter; a gene of interest; the woodchuck
hepatitis virus responsive element; a tRNA amber suppressor
sequence; a U3 element with a deletion of its enhancer sequence;
the chicken beta-globin insulator; and the R and U5 sequences of
the 3' HIV LTR.
[0169] The viral construct is preferably cloned into a plasmid that
may be transfected into a packaging cell line. The preferred
plasmid preferably comprises sequences useful for replication of
the plasmid in bacteria.
Delivery of the Virus
[0170] The virus may be delivered to the cell in any way that
allows the virus to contact the target cells in which delivery of
the gene of interest is desired. In preferred embodiments, a
suitable amount of virus is introduced into an animal directly (in
vivo), for example though injection into the body. In one such
embodiment, the viral particles are injected into the animal's
peripheral blood stream. Other injection locations also are
suitable, such as directly into organs comprising target cells. For
example intracranial or intrahepatic injection may be used to
deliver virus to the brain and liver respectively. Depending on the
particular circumstances and nature of the target cells,
introduction can be carried out through other means including for
example, inhalation, or direct contact with epithelial tissues, for
example those in the eye, mouth or skin.
[0171] In other embodiments, target cells are preferably contacted
with the virus in vitro, such as in culture plates. The virus may
be suspended in media and added to the wells of a culture plate,
tube or other container. The media containing the virus may be
added prior to the plating of the cells or after the cells have
been plated. Preferably cells are incubated in an appropriate
amount of media to provide viability and to allow for suitable
concentrations of virus in the media such that infection of the
host cell occurs.
[0172] The cells are preferably incubated with the virus for a
sufficient amount of time to allow the virus to infect the cells.
Preferably the cells are incubated with virus for at least 1 hour,
more preferably at least 5 hours and even more preferably at least
10 hours.
[0173] In both in vivo and in vitro delivery embodiments, any
concentration of virus that is sufficient to infect the desired
target cells may be used, as can be readily determined by the
skilled artisan. When the target cell is to be cultured, the
concentration of the viral particles is at least 1 pfu/.mu.l, more
preferably at least 10 pfu/.mu.l, even more preferably at least 400
pfu/.mu.l and even more preferably at least 1.times.10.sup.4
pfu/.mu.l.
[0174] In some embodiments, following infection with the virus in
vitro, target cells can be introduced into an animal. The location
of introduction of cultured cells will depend on the cell type used
and the desired effect. For example, when the cells are
hematopoietic cells, the cells can be introduced into the
peripheral blood stream. The cells introduced into an animal are
preferably cells derived from that animal, to avoid an adverse
immune response. Cells also can be used that are derived from a
donor animal having a similar immune makeup. Other cells also can
be used, including those designed to avoid an immunogenic
response.
[0175] The cells and animals incorporating target cells may be
analyzed, for example for integration, transcription and/or
expression of the gene(s) of interest, the number of copies of the
gene integrated, and the location of the integration. Such analysis
may be carried out at any time and may be carried out by any
methods known in the art.
[0176] The methods of infecting cells disclosed above do not depend
upon species-specific characteristics of the cells. As a result,
they are readily extended to all mammalian species. In some
embodiments the recombinant virus is delivered to a human or to
human cells. In other embodiments it is delivered to an animal
other than a human or non-human cells.
[0177] As discussed above, the modified retrovirus can be
pseudotyped to confer upon it a broad host range. One of skill in
the art would also be aware of appropriate internal promoters to
achieve the desired expression of a gene of interest in a
particular animal species. Thus, one of skill in the art will be
able to modify the method of infecting cells derived from any
species.
Target Cells
[0178] A wide variety of cells may be targeted in order to deliver
a gene of interest using a recombinant retrovirus as disclosed
herein. The target cells will generally be chosen based upon the
gene of interest and the desired effect.
[0179] In some embodiments, a gene of interest may be delivered to
enable a target cell to produce a protein that makes up for a
deficiency in an organism, such as an enzymatic deficiency, or
immune deficiency, such as X-linked severe combined
immunodeficiency. Thus, in some embodiments, cells that would
normally produce the protein in the animal are targeted. In other
embodiments, cells in the area in which a protein would be most
beneficial are targeted.
[0180] In other embodiments, a gene of interest, such as a gene
encoding an siRNA, may inhibit expression of a particular gene in a
target cell. The gene of interest may, for example, inhibit
expression of a gene involved in a pathogen life cycle. Thus cells
susceptible to infection from the pathogen or infected with the
pathogen may be targeted. In other embodiments, a gene of interest
may inhibit expression of a gene that is responsible for production
of a toxin in a target cell.
[0181] In other embodiments a gene of interest may encode a toxic
protein that kills cells in which it is expressed. In this case,
tumor cells or other unwanted cells may be targeted.
[0182] In still other embodiments a gene that encodes a protein to
be collected, such as a therapeutic protein may be used and cells
that are able to produce and secrete the protein are targeted.
[0183] Once a particular population of target cells is identified
in which expression of a gene of interest is desired, a target
molecule is selected that is specifically expressed on that
population of target cells. The target molecule may be expressed
exclusively on that population of cells or to a greater extent on
that population of cells than on other populations of cells. The
more specific the expression, the more specifically gene delivery
can be directed to the target cells. Depending on the context, the
desired amount of specificity of the marker (and thus of the gene
delivery) may vary. For example, for introduction of a toxic gene,
a high specificity is most preferred to avoid killing non-targeted
cells. For expression of a protein for harvest, or expression of a
secreted product where a global impact is desired, less marker
specificity may be needed.
[0184] As discussed above, the target molecule may be any molecule
for which a specific binding partner can be identified or created.
Preferably the target molecule is a peptide or polypeptide, such as
a receptor. However, in other embodiments the target molecule may
be a carbohydrate or other molecule that can be recognized by a
binding partner. If a binding partner for the target molecule is
already known, it may be used as the affinity molecule. However, if
a binding molecule is not known, antibodies to the target molecule
may be generated using standard procedures. The antibodies can then
be used as the affinity molecule, or to create an affinity
molecule.
[0185] Thus, target cells may be chosen based on a variety of
factors, including, for example, (1) the particular application
(e.g., therapy, expression of a protein to be collected, and
conferring disease resistance) and (2) expression of a marker with
the desired amount of specificity.
[0186] Target cells are not limited in any way and include both
germline cells and cell lines and somatic cells and cell lines.
Target cells can be stem cells derived from either origin. When the
target cells are germline cells, the target cells are preferably
selected from the group consisting of single-cell embryos and
embryonic stem cells (ES).
[0187] In one embodiment, target cells are CD20+ cell (see Examples
1-5). Some other non-limiting examples of target cells are CD34+
cells, CD4+ cells, dendritic cells, tumor cells and other
dysfunctional cells, and cells that are susceptible to infection
with a pathogen. Various affinity molecules are available to target
numerous cell types, for example, CD34+ cells, and dendritic cells,
described in more detail below.
[0188] Depending on the vector that is to be used, target cell
division may be required for transformation. Target cells can be
stimulated to divide in vitro by any method known in the art. For
example, hematopoietic stem cells can be cultured in the presence
of one or more growth factors, such as IL-3, IL-6 and/or stem cell
factor (SCF).
[0189] Although examples are discussed below in relation to the
targeting CD34+ stem cells and dendritic cells, one of skill in the
art will be able to adapt the disclosure to other contexts.
Targeting of Lentiviral Vectors to Human CD34+ Hematopoietic Stem
Cells
[0190] CD34 is a human hematopoietic stem cell (HSC) marker. HSCs
may be programmed to differentiate into antigen-specific immune
cells via virus-mediated gene transfer. Targeted gene delivery into
CD34+ HSC allows gene transfer in vivo, but could also be used in
vitro. Therefore, a wide range of hematological disorders, such as
various anemias, leukemias, lymphomas, and platelet disorders may
be treated using the disclosed methods and vectors to deliver the
appropriate polynucleotides to HSCs. Polynucleotides encoding
proteins whose expression in HSCs would be beneficial to treatment
of the various hematological disorders will be apparent to the
skilled artisan.
[0191] In some embodiments of the invention, CD34 is targeted.
Various examples of anti-CD34 antibodies are available and can be
used as the basis of an affinity molecule or as the affinity
molecule if they are membrane associated. For example, ATCC number
HB-12346 is readily available from the ATCC and can be used to
prepare an affinity molecule. Evaluation of the functional
expression of anti-CD34 antibodies as the affinity molecule on
recombinant retrovirus may be accomplished using a virus-cell
binding assay. TF-1a, a human CD34+ cell line, may be obtained from
the ATCC (ATCC number: CRL-2451) and used as a target cell for such
binding experiments. Transduction experiments may then be conducted
in the TF-1a line and primary human bone marrow or cord blood
cells. Transduction efficiency may be compared between various
antibodies, including natural and single chain forms. Once an
efficient affinity molecule is identified, it can be used in
conjunction with an FM, such as SINmu or HAmu, to deliver a gene of
interest to CD34+ cells, either in vivo or in vitro.
Targeting of Recombinant Virus to Dendritic Cells in vivo
[0192] Dendritic cells (DCs) have been widely used to induce
tumor-specific killer (CD8) and helper (CD4) T cell responses in
animal-tumor models and in cancer patients (Schuler et al. Curr.
Top. Microbiol. 281, 137-178 (2003)). Targeting of antigens and the
induction of their maturation are part of an in situ DC vaccination
approach. Thus, targeting lentiviral vectors to DCs in vivo using
viruses as described herein can be used therapeutically, for
example, in treating melanoma and HIV. The DEC-205 endocytosis
receptor is a preferred target surface antigen on the DC as DEC-205
is abundantly expressed on lymphoid tissue and can significantly
improve the efficiency of antigen presentation. (Bonifaz, L. C. et
al. J Exp. Med. 199, 815-824 (2004)). Anti-DEC-205 antibody,
preferably comprising the variable regions from an anti-DEC205
antibody with the constant regions of IgG1 can be displayed on the
surface of a viral particle as the affinity molecule to direct
co-delivery of both a gene encoding an antigenic protein and a
maturation stimulatory molecule, such as TNF.alpha. or CD40L, into
DCs. A maturation signal could also be delivered along with an
antigen gene by using anti-CD40 antibody as an affinity
molecule.
[0193] As described in Example 6 below, recombinant lentiviruses
have been created that co-display .alpha.mDEC-205 and SINmu to
target bone marrow derived DCs in vitro. To target human DCs,
membrane-bound antibodies against human DCs, such as IgG.sub.1
comprising the variable region from a human anti-DEC-205 antibody,
are engineered into recombinant viruses Diseases such as HIV and
melanoma can then be prevented and/or treated by delivery of
appropriate antigen genes to DCs using the recombinant virus. In
one embodiment, an anti-DEC-205 antibody comprising the variable
regions from a murine anti-DEC-205 antibody and the constant
regions from human IgG.sub.1 is used as an affinity molecule and
SINmu is used as a fusogenic molecule (see Example 6) to target a
recombinant lentivirus to DCs and deliver a gene encoding an
antigen.
Transgenic Animals
[0194] The methods of the present invention can be used to create
transgenic animals. In some embodiments particular cells in an
adult animal are targeted to deliver a polynucleotide encoding a
gene to be expressed in those cells. In other embodiments an oocyte
or one or more embryonic cells are infected with recombinant virus
produced as described above. The virus delivers a polynucleotide
encoding a gene of interest that is incorporated into the genome of
the developing animal and can be transmitted from generation to
generation. One of skill in the art will recognize that the method
of infection and the treatment of the cell following infection will
depend upon the type of animal from which the cell is obtained, and
that the ability to target gene delivery to particular cell types
allows for in vivo or in vitro gene delivery.
Therapy
[0195] The methods of the present invention can be used to prevent
or treat a wide variety of diseases or disorders. Diseases or
disorders that are amenable to treatment or prevention by the
methods of the present invention include, without limitation,
cancers, autoimmune diseases, and infections, including viral,
bacterial, fungal and parasitic infections. In some embodiments a
disease is treated by using recombinant retroviruses to deliver a
gene of interest to target cells, wherein expression of the gene
produces a protein or other molecule that addresses a deficiency in
the cell or in the animal as a whole.
[0196] In other embodiments, a gene is delivered to the target cell
type that regulates expression of a protein that is involved in the
disease or disorder. For example, a gene encoding an siRNA molecule
may be provided to inhibit a gene involved in a pathogen life
cycle, to reduce the expression of a protein that is being
overproduced, or to interfere with a pathway involved in the
progression of the disease or disorder. In other examples, genes
are delivered that inhibit a particular cellular activity by
competing with a native molecule or that enhance a cellular
activity by acting synergistically or facilitating the activity of
a native molecule.
[0197] In still other embodiments a gene is delivered that causes
the death of undesirable target cells, such as tumor cells.
Alternatively a gene may be delivered that prevents or reduces the
ability of a tumor cell to multiply.
[0198] Although illustrated in several particular contexts below,
the skilled artisan will be able to adapt the methods and
constructs disclosed herein in view of specific circumstances.
siRNAs
[0199] The methods described herein allow for vector-mediated
delivery of RNA molecules, and are particularly suited to the
delivery to and expression of small RNA molecules in target cells.
According to some embodiments of the invention, an RNA molecule is
delivered to a target cell, and then expressed within the target
cell in order to down-regulate the expression of a target gene. The
ability to down-regulate a target gene has many therapeutic and
research applications, including identifying the biological
functions of particular genes. By delivering an RNA molecule to a
target cell and subsequently expressing the RNA molecule within the
target cell, it is possible to knock-down (or down-regulate) the
expression of any of a large number of genes, both in cell culture
and in mammalian organisms. In some embodiments genes that are
necessary for the life cycle of a pathogen, such as a pathogenic
virus, or that are contributing directly or indirectly to a disease
or disorder are downregulated in a target cell with siRNA.
[0200] Thus, in some embodiments, the viral vector comprises an RNA
expression cassette encoding an siRNA molecule. An RNA expression
cassette to be delivered to a target cell preferably comprises a
Pol III promoter and an RNA coding region. The RNA coding region
preferably encodes an RNA molecule that is capable of
down-regulating the expression of a particular gene or genes. The
RNA molecule encoded can, for example, be complementary to the
sequence of an RNA molecule encoding a gene to be down-regulated.
In such an embodiment, the RNA molecule is designed to act through
an antisense mechanism.
[0201] A preferred embodiment involves the delivery to a target
cell and subsequent expression of a double-stranded RNA complex, or
an RNA molecule having a stem-loop or a so-called "hairpin"
structure. As used herein, the term "RNA duplex" refers to the
double stranded regions of both the RNA complex and the
double-stranded region of the hairpin or stem-lop structure. An RNA
coding region can encode a single stranded RNA, two or more
complementary single stranded RNAs or a hairpin forming RNA.
[0202] Double stranded RNA has been shown to inhibit gene
expression of genes having a complementary sequence through a
process termed RNA interference or suppression (see, for example,
Hammond et al. Nat. Rev. Genet. 2:110-119 (2001)).
[0203] According to some embodiments of the invention, the RNA
duplex or siRNA corresponding to a region of a gene to be
down-regulated is delivered to a target cell using the vectors
described, and is then expressed in the target cell. The RNA duplex
is substantially identical (typically at least about 80% identical,
and more typically at least about 90% identical) in sequence to the
sequence of the gene targeted for down regulation. siRNA duplexes
are described, for example, in Bummelkamp et al. Science
296:550-553 (2202), Caplen et al. Proc. Natl. Acad. Sci. USA
98:9742-9747 (2001) and Paddison et al. Genes & Devel.
16:948-958 (2002).
[0204] The RNA duplex to be delivered to the target cell is
generally at least about 15 nucleotides in length and is preferably
about 15 to about 30 nucleotides in length. In some organisms, the
RNA duplex can be significantly longer. In a more preferred
embodiment, the RNA duplex is between about 19 and 22 nucleotides
in length. The RNA duplex is most preferably identical to the
target nucleotide sequence over the duplex region.
[0205] When the target cell gene to be down regulated is in a
family of highly conserved genes, the sequence of the duplex region
can be chosen with the aid of sequence comparison to target only
the desired gene. If there is sufficient identity among a family of
homologous genes within an organism, a duplex region can be
designed that would down regulate a plurality of genes
simultaneously.
[0206] The sequence of the RNA coding region, and thus the sequence
of the RNA duplex to be delivered to the target cell, preferably is
chosen to be complementary to the sequence of a gene whose
expression is to be downregulated in a target cell. The degree of
down regulation achieved with a given RNA molecule for a given
target gene will vary by sequence. One of skill in the art will be
able to readily identify an effective sequence. For example, in
order to maximize the amount of suppression, a number of sequences
can be tested in cell culture prior to treating target cells.
[0207] In some embodiments, the target (within the target cell) of
the RNA duplex is a sequence that is necessary for the life cycle
or replication of a virus, including for example, gene expression
of the virus and the expression of a cellular receptor or
co-receptor necessary for viral replication. In one embodiment of
the invention, the virus to be inhibited is the human
immunodeficiency virus (HIV).
[0208] In some embodiments, the gene of interest to be delivered to
the target cell encodes at least one double stranded RNA having at
least 90% homology and preferably identical to a region of at least
about 15 to 25 nucleotides in a nucleotide that is important for
normal viral replication. For example, the double stranded RNA may
have homology to a nucleic acid in a viral genome, a viral gene
transcript or in a gene for a patient's target cellular receptor
that is necessary for the life cycle of the virus.
[0209] In some embodiments, siRNAs are delivered to treat infection
such as HIV, Hepatitis A, B, C, D, E, or a wide range of other
viral infections. One of skill in the art can target a cellular
component, either an RNA or an RNA encoding a cellular protein
necessary for a pathogen life cycle, such as a viral life cycle. In
a preferred embodiment, the cellular target chosen will not be a
protein or RNA that is necessary for normal cell growth and
viability. Suitable proteins for disrupting the viral life cycle
include, for example, cell surface receptors involved in viral
entry, including both primary receptors and secondary receptors,
and transcription factors involved in the transcription of a viral
genome, proteins involved in integration into a host chromosome,
and proteins involved in translational or other regulation of viral
gene expression.
[0210] A wide variety of molecules are specifically associated with
pathogens and can be targeted by the methods disclosed herein.
These include a number of cellular proteins that are known to be
receptors for viral entry into cells (Baranowski, et al. Science
292: 1102-1105). Some cellular receptors that are involved in
recognition by viruses are listed below: Adenoviruses: CAR,
Integrins, MHC I, Heparan sulfate glycoaminoglycan, Siliac Acid;
Cytomegalovirus: Heparan sulfate glycoaminoglycan;
Coxsackieviruses: Integrins, ICAM-1, CAR, MHC I; Hepatitis A:
murine-like class I integral membrane clycoprotein; Hepatitis C:
CD81, Low density lipoprotein receptor; HIV (Retroviridae): CD4,
CXCR4, Heparan sulfate glycoaminoglycan; HSV: Heparan sulfate
glycoaminoglycan, PVR, HveB, HveC; Influenza Virus: Sialic acid;
Measles: CD46, CD55; Poliovirus,: PVR, HveB, HveC; Human
papillomavirus: Integrins. One of skill in the art will recognize
that the invention is not limited to use with receptors (or other
molecules) that are currently known. As new cellular receptors and
coreceptors are discovered, the methods of the invention can be
applied to such sequences.
[0211] In some embodiments of the invention, HIV is particularly
targeted and the retroviral construct comprises an RNA coding
region that encodes a double stranded molecule having at least 90%
homology to the HIV viral RNA genome, an expressed region of the
HIV viral genome (for example, to any region of about 19-25
nucleotides in length of the 9-kb transcript of the integrated HIV
virus), or any of the variously spliced mRNA transcripts of HIV
(Schwartz et al. J Virol. 1990; 64(6): 2519-29). Target regions
within the HIV transcripts can be chosen to correspond to any of
the viral genes, including, for example, HIV-1 LTR, vif, nef, and
rev. In other embodiment,s the RNA coding region encodes a double
stranded region having at least 90% homology to a receptor or
co-receptor of the HIV virus. For example, the primary receptor for
HIV entry into T cells is CD4. In a preferred embodiment, the
co-receptors CXC chemokine receptor 4 (CXCR4) and CC chemokine
receptor 5 (CCR5) are down-regulated according to the methods of
the invention. CXCR4 (Feddersppiel et al. Genomics 16:707-712
(1993)) is the major co-receptor for T cell trophic strains of HIV
while CCR5 (Mummidi et al. J. Biol. Chem. 272:30662-30671 (1997))
is the major co-receptor for macrophage trophic strains of HIV.
Other cellular targets against HIV include the RNA transcripts for
proteins involved in the HIV life cycle, including cyclophilin,
CRM-1, importin-13, HP68 (Zimmerman C, et al. Identification of a
host protein essential for assembly of immature HIV-1 capsids.
Nature 415 (6867): 88-92 (2002)) and other as yet unknown cellular
factors.
[0212] In one particular embodiment, a recombinant retrovirus is
used to introduce siRNAs against the HIV-1 co-receptor CCR5 into
human peripheral blood T cells. Reducing CCR5 expression by siRNAs
provides protection from CCR5-tropic HIV-1 viral infection (Qin et
al. (2003). Proc. Natl. Acad. Sci. 100, 183-188). Targeted delivery
of such siRNAs to human CD34+ cells may thus reconstitute CD4+
cells that are resistant to HIV-1 infection. Recombinant
lentiviruses comprising an affinity molecule that targets CD34,
such as an anti-CD 34 antibody, a fusogenic molecule such as SIN or
HA, and encoding CCR5-siRNA and, optionally, GFP may be injected
intravenously to treat HIV.
Vaccination
[0213] As discussed above, various cell-specific binding
determinants to surface dendritic cell markers are contemplated for
use in producing recombinant retrovirus that delivers a gene
encoding an antigen to DCs. For example, a hybridoma cell line for
human anti-DEC-205 antibody (.alpha.hDEC-205) is available from the
ATCC (ATCC number: CRL-2460). A gene encoding an antigen against
which an immune response is desired, such as for cancer (for
example, Mart-1), or another disease/disorder (such as viral
infection) may be delivered to DCs using the methods described
above. The gene for the antigen may be accompanied by genes
encoding stimulatory molecules, such as TNF.alpha./CD40L, and/or a
reporter molecule, such as GFP using multiple vectors or,
preferably, a multicistronic vector system.
[0214] In some embodiments of the invention, human DCs are
generated from CD34.alpha.+ human hematopoietic progenitors using
an in vitro culture method (e.g., Banchereau et al. Cell 106,
271-274 (2001)). .alpha.hDEC-205 and SINmu-bearing viruses are
generated comprising a gene encoding an antigen against which an
immune response is desired and are used to transduce human DCs.
Transduction specificity and efficiency may be quantified by FACS.
Maturation of DCs can be characterized by FACS analysis of
up-regulation of surface marker such as MHC II.
[0215] In other embodiments, virus may be injected in vivo, where
it contacts natural DCs and delivers the gene encoding the antigen.
At selected intervals, DCs from the recipient's lymphoid organs may
be used to measure expression, for example, by observing marker
expression, such as GFP. T cells from lymph nodes and spleens of
virus-treated recipients may be measured from the magnitude and
durability of response to antigen stimulation. Tissue cells other
than DCs, such as epithelial cells and lymphoid cells, may be
analyzed for the specificity of in vivo gene delivery.
[0216] It is widely agreed that the most effective potential method
to end the AIDS epidemic (and other viral diseases) is a vaccine.
Unfortunately, to date no vaccination method against HIV has
successfully passed a phase III trial. Thus, there is an urgent
need for novel and effective vaccination strategies. In some
embodiments of the invention DC vaccination is used. A gene is
cloned encoding a viral protein, such as those described above,
into a viral vector. Patients are infected with viruses comprising
an affinity molecule that targets DCs, such as .alpha.hDEC-205 by
injection. In an animal model, molecularly cloned HIV reporter
viruses (NFNSZ-r-HSAS, NL-r-HSAS) and clinical isolates may be used
to challenge the animals by tail vein injection. Evidence of
infection may be monitored over time in splenocytes, lymph nodes,
and peripheral blood. PCR for HIV-gag protein and FACS for HAS in
the reporter viruses may be used to test for viral integration and
replication. Productive in situ DC vaccination may increase
resistance to a HIV challenge.
Treatment of Tumors and Other Abnormal Cells
[0217] In other embodiments, the disclosed method can be used to
treat tumors or other abnormal cell growth. Tumor associated
antigens are known for a variety of cancers including, for example,
prostate cancer and breast cancer. In some breast cancers, for
example, the Her-2 receptor is overexpressed on the surface of
cancerous cells. A number of tumor associated antigens have been
reviewed (see, for example, Boon T, Cerottini J C, Vandeneynde B,
Vanderbruggen P, Vanpel A, Annual Review Of Immunology 12: 337-365,
1994; Renkvist N, Castelli C, Robbins P F, Parmiani G. Cancer
Immunology Immunotherapy 50: (1) 3-15 MAR 2001). Thus, in some
embodiments an antibody to a known tumor associated antigen is used
to prepare an affinity molecule.
[0218] In other embodiments, an antigen related to a disease or
disorder is identified from the patient to be treated. For example,
an antigen associated with a tumor may be identified from the tumor
itself by any method known in the art.
[0219] Antibodies to tumor associated antigens may be displayed on
a viral surface to target delivery of genes of interest into tumor
cells. The genes of interest may encode a toxin whose expression
kills the target tumor cells. In some embodiments the expression of
the toxin is inducible. In other embodiments the gene of interest
may interfere with the cell cycle and reduce or eliminate the
ability of the cell to divide.
[0220] These methods may be adapted to treat a wide range of
diseases by selecting a particular target molecule on a pathologic
cell of interest and synthesizing an affinity molecule to that
particular target molecule. Next, a recombinant retrovirus with the
membrane bound affinity molecule and a fusogenic molecule is
assembled to deliver the gene of interest into the target cell.
[0221] In some embodiments, a recombinant retrovirus may be used to
target a non-Hodgkin's lymphoma cell. A HSV thymidine kinase
(HSV-tk) suicide gene and a GFP reporter gene may be delivered into
tumor cells; these two genes may be linked by an internal ribosome
entry site (IRES) to accomplish co-expression.
[0222] In some embodiments, a retrovirus as described herein may be
used to target a tumor cell with a specific cell surface antigen,
such as a breast cancer tumor cell. A hybridoma cell line for
anti-human Her2 antibody is available from the ATCC (ATCC number:
CRL: 1043). Viruses bearing such antibodies may be used to target
and kill cancer cells.
Treatment of X-linked Severe Combined Deficiency
[0223] Genetic defects in the common .beta. chain (.beta..sub.c)
result in the X-linked severe combined immunodeficiency disease
(X-SCID) in humans. Without treatment, X-SCID patients suffer from
severe infections, failure to thrive, and usually die within the
first year of life. It is commonly agreed that the ultimate
therapeutic treatment for this disease is gene therapy. A
retrovirus as disclosed may be used to deliver the common
.beta..sub.c gene into purified CD34+ hematopoietic stem cells
(HSCs) in vitro and transfer back .beta..sub.c-transduced HSCs to
patients to reconstitute the immune system. In other embodiments,
the recombinant virus is provided in vivo and targets CD34+ stem
cells to treat X-SCID. SCF can be used to specifically target the
recombinant retrovirus to the target cells, where the .beta..sub.c
gene is delivered.
[0224] In one embodiment patients suffering from X-SCID are
treated. The full length of .beta..sub.c cDNA is amplified and
cloned into a lentiviral vector as described above. Packaging
cells, such as 293 cells are transfected with the lentiviral
vector, as well as one or more vectors encoding an affinity
molecule and a fusogenic molecule. The viruses bearing SCF, or
another affinity molecule targeting HSCs and a fusogenic molecule,
such as SINmu, are collected and concentrated. The
.beta..sub.c-deficient patients are administered the viruses by
injection. Testing with and without mobilization may be performed
to relocate HSCs to circulating blood. Peripheral lymphoid cells
may then be analyzed for 6-8 weeks to detect the existence of
mature T and B cells.
Antigen-Specific Immune Cell Therapy
[0225] In other embodiments recombinant retrovirus is used to
deliver polynucleotides encoding immune cell receptors, such as T
Cell receptors or B cell receptors, to human stem cells. The stem
cells then develop into mature immune cells, such as T cells or B
cells, with their specificity determined by the receptor with which
they were transduced. In one embodiment, a patient that is
suffering from a disease or disorder is treated by generating
immune cells with a desired specificity using this approach. An
antigen may be previously known to be associated with the disease
or disorder, or may be identified by any method known in the art.
For example, an antigen to a type of cancer from which a patient is
suffering may be known, such as a tumor associated antigen. Tumor
associated antigens are not limited in any way and include, for
example, antigens that are identified on cancerous cells from the
patient to be treated.
[0226] Once an antigen has been identified and/or selected, one or
more T cell receptors that are specific for the antigen are then
identified. If a T cell receptor specific for the identified
disease-associated antigen is not already known, it may be
identified by any method known in the art. T cell receptors may be
identified from cytotoxic T cells, from helper T cells, or both,
depending on the type of immune cell that is to be generated in the
patient. For example, if cytotoxic T cells are to be generated in
the patient, the T cell receptor is identified from a CTL. On the
other hand, if helper T cells are to be generated, the T cell
receptor is identified from a helper T cell. As discussed below, in
some embodiments a T cell receptor from a CTL and a T cell receptor
from a helper T cell are both utilized.
[0227] A polynucleotide that encodes the desired T cell receptor is
identified. Preferably the polynucleotide comprises a cDNA that
encodes the T cell receptor .alpha. subunit and a cDNA that encodes
the T cell receptor .beta. subunit. The polynucleotides encoding
the T cell receptor are preferably introduced into target cells
(preferably hematopoietic stem cells) using a modified retrovirus,
more preferably a modified lentivirus, including a fusion molecule
and cell-specific binding determinant as described above. The virus
first binds to the target cell membrane by way of the
membrane-bound affinity molecule, and the polynucleotides encoding
the T cell receptor subunits enter the cytosol by action of the
fusion molecule. The gene of interest (e.g., one encoding the T
cell receptor) is then preferably integrated into the cell's genome
and expressed. If contacted ex vivo, the target cells are then
transferred back to the patient, for example by injection, where
they develop into immune cells that are capable of generating an
immune response when contacted with the identified antigen.
However, in preferred embodiments the virus is injected into the
patient where it specifically transduces the targeted cells. The
resulting immune cells generated in the patient express the
particular TCR and the patient is able to mount an effective immune
response against the disease or disorder.
[0228] In some embodiments the T cell receptor is cloned from
cytotoxic T cells. This results in the generation of cytotoxic T
cells in the patient. In other embodiments the T cell receptor is
cloned from a helper T cell, resulting in the generation of helper
T cells in the patient.
[0229] In still other embodiments B cells are generated in the
patient by delivering polynucleotides encoding B cell receptors to
the target cells. The population of target cells is divided and
some stem cells are transfected with a vector encoding a T cell
receptor obtained from a cytotoxic T cell and some stem cells are
transfected with a vector encoding a T cell receptor obtained from
a helper T cell. The target stem cells are transferred into the
patient, resulting in the simultaneous generation of a population
of helper T cells specific for the disease or disorder and a
population of cytotoxic T cells specific for the disease or
disorder in the patient.
[0230] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
[0231] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
Experimental Methods
[0232] The following experimental methods were used in Examples 1-5
below.
Construct Preparation
[0233] The cDNAs of the light and heavy chain constant regions of
the membrane bound human IgG.sub.1 were amplified and inserted
downstream of human CMV and EF1.alpha. promoters, respectively, of
the pBudCE4.1 vector (Invitrogene). The light and heavy chain
variable regions form the murine anti-CD20 antibody (clone 2H7)
were then cloned using PCR amplification and inserted directly
upstream of the corresponding constant regions. The resulting
construct was designated as p.alpha.CD20. cDNAs of human Ig.beta.
and Ig.beta. were also cloned into a pBudCE4.1 vector (Invitrogene)
to yield pIg.alpha..beta..
[0234] The construct encoding HAmu was provided by the laboratory
of Dr. Cannon at the University of Southern California (A. H. Lin
et al., Hum. Gene. Ther. 12, 323 (2001)). The cDNA for wild-type
SIN was obtained from Dr. Strauss's laboratory at Caltech. PCR
mutagenesis and assembly were used to generate the mutant SIN as
described in Morizono et al., Nature Med. 11, 346 (2005), except
that a 10 amino acid residue tag sequence replaced the ZZ domain of
protein A, which is located between amino acid 71 and 74 of the E2
glycoprotein of SIN. This version of SIN is designated as
SINmu.
Virus Production
[0235] Lentivirus were generated by transfecting 293T cells using a
standard calcium phosphate precipitation technique. 293T cells
(.about.80% confluent) in 6-cm culture dishes were transfected with
the appropriate lentiviral vector plasmid (5 .mu.g), together with
2.5 .mu.g each of p.alpha.CD20, pIg.alpha..beta., and the package
vector plasmids (pMDLg/pRRE and pRSV-Rev) (Sandrin et al. Curr.
Top. Microbio. Immunol. 281:137 (2003)). The viral supernatants
were harvested 48 and 72 hours after transfection and filtered
through a 0.45-.mu.m pore size filter.
[0236] To prepare high titer lentivirus, the viral supernatants
were concentrated using ultracentrifugation (Optima L-80 K
preparative ultracentrifuge, Beckman Coulter) for 90 min at
50,000.times.g. Particles were then resuspended in an appropriate
volume of cold PBS.
Cell Line Construction
[0237] The 293T/CD20 cell line was generated by stable transduction
via VSVG-pseudotyped lentivirus. The cDNA of human CD20 was cloned
downstream of the human ubiquitin-C promoter in the plasmid FUW
(FUGW without GFP; Lois et al. Science 295:868-872 (2002)) to
generate FUW-CD20. The lentiviral vector FUW-CD20 was then
pseudotyped with VsVg and was used to transduce 293T. The resulting
cells were subjected to cell sorting to obtain a uniform population
of CD20+ cells designated as 293T/CD20.
Virus-cell Binding Assay
[0238] Cells (293T/CD20 or 293T, 0.1.times.10.sup.6) were incubated
with 500 .mu.L of viral supernatant at 4.degree. C. for half an
hour and washed with 4 ml of cold PBS. The cells were then stained
with the following three antibodies: an anti-human IgG antibody (BD
Pharmingen) to stain .alpha.CD20, an anti-human CD20 antibody (BD
Pharmingen) to stain CD20, and an anti-FPV HA polyclonal antibody
(obtained from H.-D. Klenck, Institute of Virology;
Philipps-University, Marburg, Germany) to stain HAmu, or an
anti-tag antibody (Roche) to stain SINmu. After staining, cells
were analyzed by fluorescence-activated cell sorting (FACS.
Targeted Transduction of 293T/CD20 Cells in vitro
[0239] 293T/CD20 cells (0.2.times.10.sup.6/well) or 293T cells
(0.2.times.10.sup.6/well) were plated in a 24-well culture dish,
and spin-infected with viral supernatants (0.5 ml/well) at 2,500
rpm, 30.degree. C. for 90 min. using a Beckman Allegra 6R
centrifuge. Then the medium was removed and replaced with fresh
medium and incubated for a further 3 days at 37.degree. C. with 5%
CO.sub.2. The percentage of GFP.sup.+ cells was determined by FACS.
The transduction titer was measured at the dilution ranges that
exhibited a linear response.
Effects of Soluble Antibody and NH4Cl on Viral Transduction
[0240] 293T/CD20 cells (0.1.times.10.sup.6) and 0.5 mL of viral
supernatants were incubated for 8 hours in the absence or presence
of a gradient dose of anti-human CD20 antibody (BD Pharmingen) or
NH.sub.4Cl. The medium was replaced with fresh medium and incubated
for another 2 days at 37.degree. C. with 5% CO.sub.2. FACS analysis
was used to quantify transduction efficiency.
Cell-cell Fusion Assay
[0241] 293T cells (0.1.times.10.sup.6) transiently transfected to
express GFP and surface .alpha.CD20 and fusion protein (either HAmu
or SINmu), and 293T/CD20 cells (0.1.times.10.sup.6) were mixed
together, washed twice with normal PBS (pH=7.4), and incubated in
150 .mu.l low pH PBS (pH-5.0) or normal pH PBS (pH=7.4) (as a
control) for half an hour at 37.degree. C. with 5% CO.sub.2. The
cells were then washed extensively and cultured in the regular
medium for one day. Cells were visualized by an epifluorescence
microscope equipped with a GFP filter set. Targeted transduction of
primary human B cells in vitro
[0242] Fresh, un-fractionated human peripheral blood mononuclear
cells (PBMCs) (0.2.times.10.sup.6) (AllCells, LLC) were incubated
with concentrated virus with total transduction units (TU) of
10.times.10.sup.6 (based on the titer on 293T/CD20 cells). LPS (50
.mu.g/mL) was then added for B cells to survive and grow. After two
days, cells were harvested and washed in PBS. B cell population was
determined by FACS staining using anti-human CD20 and CD19
antibodies. Targeting transduction was quantified by gating on the
different populations of cells and measuring their GFP
expression.
Targeted Transduction of Primary Human B Cells in vivo
[0243] RAG.sup.-/-.sub..gamma.c.sup.-/- female mice (Taconic) of
6-8 weeks old were given 360 rads whole body irradiation. On the
following day, 100.times.10.sup.6 fresh human PBMCs (AllCells, LLC)
were transferred by tail vein injection into each mouse. After six
hours, concentrated viruses (100.times.10.sup.6 TU/mouse) or PBS
(as control) were administered into these mice via the tail vein.
Two days later, whole blood was collected from these mice via heart
puncture and the cells were stained for human CD3 and CD20 and then
analyzed by FACS for CD3, CD20 and GFP expression. The mice were
maintained on the mixed antibiotic sulfmethoxazole and trimethoprim
oral suspension (Hi-Tech Pharmacal) in a sterile environment in the
California Institute of Technology animal facility in accordance
with institute regulations.
EXAMPLE 1
Targeted Cell Transduction Utilizing Retroviral Vectors
FUGW/.alpha.CCD20+HAmu and FUGW/.alpha.CD20+SINmu.
[0244] One antibody chosen to serve as the basis of an affinity
molecule, according to some embodiments of the invention, is the
anti-CD20 antibody (.alpha.CD20), a version of which is currently
being used in the treatment of B-cell lymphomas. Physiologically,
CD20 is expressed at the pre-B-cell stage of development and
throughout B-cell maturation; hematopoietic stem cells do not
express CD20. When B-cells mature into plasma cells, expression of
CD20 is diminished. Thus, CD 20 represents an ideal target for
therapy of, for example, B-cell lymphomas and leukemia. A construct
that encodes a mouse/human chimeric anti-CD20 antibody with the
human membrane-bound IgG constant region (p.alpha.CD20) was
generated as described above. Genes encoding human Ig.alpha. and
Ig.beta., the two associated proteins that are required for surface
expression of antibodies, were cloned into a construct designated
pIg.alpha..beta.(FIG. 1).
[0245] The production of lentiviruses enveloped with both anti-CD20
antibody and candidate fusion molecules (FMs) was achieved by
co-transfection of 293T cells with the lentiviral vector FUGW (Lois
et al. Science 295:868-872 (2002)), plasmids encoding viral gag,
pol, and rev genes, p.alpha.CD20, pIg.alpha..beta. and pFM (the
plasmid encoding a FM, either HAmu or SINmu), using a standard
calcium phosphate precipitation method. FUGW is a self-inactivating
and replication-incompetent lentiviral vector which carries the
human ubiquitin-C promoter driving the GFP reporter gene (C. Lois,
E. J. Hong, S. Pease, E. J. Brown, D. Baltimore, Science 295, 868
(2002)). As a control, the envelope glycoprotein derived from
vesicular stomatitis virus (VSVG) was used as recognition and
fusion protein.
[0246] FACS analysis of the transfected cells showed that virtually
all expressed some level of GFP as an indicator of the presence of
the viral vector (FIGS. 3B and 3D, upper panels). Some 30% of
GFP-positive cells co-expressed HAmu and .alpha.CD20 on the cell
surface (FIG. 3B, lower panel). A slightly smaller percentage
(.about.20%) of the 293T cells exhibited co-expression of GFP,
SINmu, and .alpha.CD20 (FIG. 3D). The resultant viruses from these
transfected production cells were designated FUGW/60 CD20+HAmu and
FUGW/.alpha.CD20+SINmu.
[0247] To examine whether .alpha.CD20 and the FM were incorporated
in the same virion, a virus-cell binding assay was performed. As a
target, a 293T cell line was made stably expressing the CD20
protein antigen, as described above (293T/CD20, FIG. 4A). The
parental cell line 293T served as a negative control. The viral
supernatants were incubated with the target cells at 4C for half an
hour. The resultant binding was assayed via a three-staining scheme
(FIG. 4B). FACS analysis showed that recombinant lentivirus bearing
.alpha.CD20 was in fact able to bind to CD20 expressing 293T cells
(FIG. 4C, upper panels). The control of 293T cells with no CD20
expression displayed no detectable .alpha.CD20, showing that the
virus binding to cells must be due to a specific interaction
between the cell surface CD20 antigen and the viral surface
.alpha.CD20 molecule. In another control, the virus bearing only FM
exhibited no ability to bind both cells, indicating that the HAmu
and SINmu did lack the capacity for cell binding. FACS analysis
also showed that the virus bound to the 293T/CD20 cell surface
displayed the FMs (FIG. 4C, lower panels), suggesting that both
.alpha.CD20 and FM were incorporated on the same virion, which was
further confirmed by FACS plots of .alpha.CD20 versus FM (FIG. 4D).
In addition to co-display, these results indicate that the presence
of the FM does not affect the .alpha.CD20 binding to CD20.
EXAMPLE 2
Transduction of CD20-expressing Target cells and 293T Cells
Utilizing the Retroviral Vector FUGW/.alpha.CD20+HAmu and FUGW/60
CD20+SINmu
[0248] Next, the efficacy of a .alpha.CD20-bearing virus in
transferring genes into cells expressing CD20 in a cell-specific
manner was tested. GFP expression was used to measure the
transduction efficiency. The supernatants containing virus bearing
various surface proteins were incubated with CD20-expressing target
cells and 293T cells served as a control. Four days
post-transduction, the efficiency of targeting was analyzed by
FACS. FIG. 5A (rightmost panel) shows that FUGW/.alpha.CD20+HAmu
viral particles could specifically transduce 16% of 293T/CD20
cells. Panels to the left show that transduction required the
presence on the virions of HAmu, but there was some background
transduction with virions lacking .alpha.CD20, likely due to
residual weak binding of HAmu to its ligand, sialic acid. The titer
for FUGW/.alpha.CD20+HAmu (fresh viral supernatant, no
concentration) was estimated to be .about.1.times.10.sup.5
transduction units (TU)/mL. The titer was determined by the
percentage of GFP+ cells in the dilution ranges that showed a
linear response. The 293T cells showed a small background infection
level but no specific transduction by FUGW/.alpha.CD20+Hamu (FIG.
5A, lower panels).
[0249] When SINmu was used as the fusion protein, substantial
enhancement of specific transduction was observed (52%, FIG. 5B).
The titer for FUGW/.alpha.CD20+SINmu was estimated to be
.about.1.times.10.sup.6 TU/mL. Also, a much lower transduction was
detected in the absence of the binding protein (.about.1%). Thus
the data in FIG. 5B shows that SINmu is a preferred fusion protein
to partner with 60 CD20 for targeting the virus. When the
transduction was monitored at various time points using FACS, it
was found that SINmu-containing virions exhibited faster
transduction kinetics than those with HAmu. Both
FUGW/.alpha.CD20+HAmu and FUGW/.alpha.CD20+SINmu could be
concentrated by ultracentrifugation with a >90% recovery rate,
which is important for in vivo applications.
[0250] To assess whether .alpha.CD20 and the fusion protein (HAmu
or SINmu) had to be incorporated into the same viral particle, and
therefore functioned in cis to mediate transduction, Virus
generated from FUGW/.alpha.CD20 was mixed with virus generated from
FUGW/HAmu or FUGW/SINmu, each displaying only one protein, and
their transduction of 293T/CD20 cells was tested. This procedure
did not result in specific transduction, indicating that the
specific transduction conferred by the engineered recombinant
viruses requires that the two proteins be displayed on the same
viral particle.
[0251] Thus, two distinct proteins can contribute to the binding
and fusion events of engineered lentiviruses for targeted
transduction. To further confirm that the specificity observed was
a consequence of interaction between .alpha.CD20 and CD20,
293T/CD20 cells were transduced in the presence of anti-CD20
blocking antibody. As expected, a dramatic decrease of infectivity
was detected for both FUGW/.alpha.CD20+HAmu and
FUGW/.alpha.CD20+SINmu virus (FIG. 5D), indicating that
antibody-antigen binding facilitates targeted transduction.
[0252] To examine the requirement for a low pH compartment to allow
the recombinant lentivirus to penetrate into cells, both
FUGW/.alpha.CD20+HAmu and FUGW/.alpha.CD20+SINmu virus was
incubated with 293T/CD20 cells in the absence or presence of
ammonium chloride (NH.sub.4Cl), which neutralizes acidic endosomal
compartments. Addition of NH.sub.4Cl to cells completely abolished
transduction by either FUGW/.alpha.CD20+HAmu virus (not shown) or
FUGW/60 CD20+SINmu (FIG. 5E). These results are consistent with the
low pH requirement of hemagglutinin and Sindbis virus glycoprotein
to trigger membrane fusion.
[0253] More direct evidence for pH dependent fusion was provided by
a cell-cell fusion assay. 293T cells expressing GFP and surface
.alpha.CD20 and FM were incubated with 293T/CD20 cells in a low-pH
buffer for half an hour, followed by culturing in regular medium.
Both HAmu and SINmu induced cell-cell fusion by forming
multi-nucleated polykaryons (FIG. 5C). The interaction between
.alpha.CD20 and CD20 dramatically enhances the probability of
fusion, because a similar experiment with cells that lacked
.alpha.CD20 and CD20 yielded a much lower level of fusion. The
.alpha.CD20/CD20 interaction brings the cell membranes into close
approximation, facilitating the action of the fusion protein.
EXAMPLE 3
Transduction of Primary Human B-Lymphoid Cells Using the Retroviral
Vector FUGW/.alpha.CD20+SINmu
[0254] Having established the ability of the system to mediate
CD20-specific transduction of artificially created cell lines, the
specific transduction of primary human B-lymphoid cells, cells that
naturally carry the CD20 antigen, was investigated. Fresh,
unfractionated human peripheral blood mononuclear cells (PBMCs)
were transduced with FUGW/.alpha.CD20+SINmu and then stimulated
with lipopolysaccharide (LPS) to expand the B cell population. Four
days later, the cells were stained for CD19 (a B cell marker), CD20
and GFP expression (FIG. 6A). Over 35% of cells were CD20+ B cells
under the described culture condition. The majority of them were
GFP+. On the contrary, virtually no GFP+ cells were detected among
CD20- non-B cells, confirming that the transduction was strictly
dependent on CD20 expression. In another control experiment, fresh
PBMCs were transduced with FUGW/.alpha.CD20+SINmu followed by
stimulation with phorbol-12-myristate-13-acetate (PMA) and
ionomycin to expand T cells. FACS analysis of these T cells showed
no expression of GFP (FIG. 7), confirming transduction
specificity.
EXAMPLE 4
Demonstration of Transduction Utilizing Lentiviral Vectors
CCMV/.alpha.CD20+SINmu and CPGK/.alpha.CD20+SINmu
[0255] To demonstrate that the targeting method is not limited to
the lentiviral vector FUGW, two additional lentiviral vectors with
different promoter configurations were evaluated. Kohn et al. have
incorporated the immunoglobulin heavy chain enhancer (E.mu.) with
associated matrix attachment regions into lentivectors carrying
either the human cytomegalovirus (CMV) promoter (CCMV) or the
murine phosphoglycerate kinase promoter (CPGK) (C. Lutzko et al. J
Virol. 77, 7341-51 (2003)). These two lentiviral vectors were then
adapted into the system and recombinant lentiviruses
CCMV/.alpha.CD20+SINmu and CPGK/.alpha.CD20+SINmu were prepared.
Transduction of PBMC-derived B cells with these viral supernatants
exhibited results similar to those observed previously with FUGW
(FIG. 6A). Stable integration of the GFP-transgene was detected by
genomic PCR amplification (FIG. 6B).
EXAMPLE 5
Testing of in vivo Efficacy of Lentiviral Vectors in Mediating
Specific Transduction
[0256] Next, the efficacy of the system in mediating specific
transduction in vivo was tested. For this purpose, a human PBMC
xenografted mouse model was used. Fresh human PBMCs
(100.times.10.sup.6/mouse) were transferred into irradiated
immunodeficient RAG2.sup.-/-.gamma..sub.c.sup.-/- mice through a
tail vein injection. Engineered lentiviruses bearing .alpha.CD20
and SINmu were administered through the tail vein 6 hours after
human cell transfer. After 2 days, whole blood from these mice was
collected and the cells were analyzed for surface antigens and GFP
expression.
[0257] Approximately 30-40% of the cells recovered from the mice
were human T cells (CD3.sup.+) and 0.1.about.0.3% were CD20.sup.+
human B cells. Three populations were analyzed for GFP expression:
CD20.sup.+, CD3.sup.+, and CD20.sup.-CD3.sup.-. None of the cells
harvested from mice injected with virus bearing a control antibody
and SINmu (FUGW/b12+SINmu) showed evidence of GFP expression in any
of the three populations (FIG. 6C). In contrast, GFP expression was
observed in at least 40% of the CD20.sup.+ cells isolated from mice
injected with FUGW/.alpha.CD20+SINmu while no transduction was
detected in the other two populations.
[0258] This demonstration of targeting efficient gene delivery
vehicles strictly to the desired cell types in vivo allows for
lentivirus-mediated gene therapy and alleviates concerns of
off-target effects. Possibly the most important implication of the
work is that gene therapy can now be carried out as an inexpensive
procedure, and thus a viable consideration even in the
less-developed world.
EXAMPLE 6
Infection of Dendritic Cells Expressing the DEC-205 Receptor by
Recombinant Lentiviral Vector FUGW/.alpha.mDEC-205+SINmu
[0259] To evaluate the use of affinity molecules such as surface
antibodies to target lentiviral vectors, membrane-bound antibody
against the mouse DEC-205 receptor (designated as .alpha.mDEC-205)
was prepared as described above for the anti-CD20 antibody.
.alpha.mDEC-205 is an endocytic receptor abundantly expressed on
dendritic cells (DCs).
[0260] A protocol was adopted to generate mouse DCs from
progenitors grown in bone marrow cultures (Yang L and Baltimore D.
Proc. Natl. Acad. Sci. USA 102:4518 (2005)). Bone marrow cells were
harvested from mice and cultured in vitro in the presence of
granulocyte-macrophage colony stimulating factor (GM-CSF). On day
10 cells were collected and it was confirmed that 70% of the cells
expressed DEC-205. Virus-cell binding assay showed that recombinant
FUGW/ .alpha.mDEC-205+SINmu could bind to DEC-205 positive cells.
When the DCs complexed with viruses were analyzed, significant
downregulation of DEC-205 was observed. Infection of these cells
with viral supernatants followed a very similar pattern to that
seen previously with .alpha.CD-20. When DCs were gated on (mCD11c
high), FUGW/ .alpha.mDEC-205+SINmu exhibited high infection
efficiency (42%) whereas FUGW/.alpha.mDEC-205 and FUGW/SINmu
exhibited virtually no infection. Downregulation of DEC-205 was
observed on DCs infected with viruses bearing .alpha.mDEC-205.
These results also showed that recombinant retroviruses such as
described above can efficiently infect primary cells.
EXAMPLE 7
Use of a Single Chain Membrane-bound Antibody (Anti-CD 20) with a
Recombinant Lentivirus to Target CD20-expressing Cells
[0261] A single chain membrane-bound form of antibody (scAbm) was
developed as an affinity molecule target recombinant lentivirus.
scAbms are typically designed to have heavy chain and light chain
variable domains linked by a flexible peptide linker. They also
carry a signal peptide at their N terminus and a transmembrane
domain at their C terminus for anchoring to the cell surface. A
slightly different version of scAbm devised is designated as
sc.alpha.CD20. This scAbm was composed of heavy chain and light
chain variable domains of anti-CD20 antibody linked by
(GGGGSGGGS).sub.2 peptide, and a dimerization region including the
hinge CH2-CH3 domain of human IgG1, and the transmembrane domain
and the cytoplasmic tail of the human HLA-A2 to display this
chimeric protein on the cell surface.
[0262] The ability of sc.alpha.CD20 to be expressed on the cell
surface was examined by FACS analysis. Transfection of 293T cells
with the expression vector of psc.alpha.CD20 resulted in higher
levels of surface antibody expression, when compared to those
versions of scAbms without a dimerization domain in the literature
(e.g., de Ines, C. et al. J. Immunol. 163, 3948-3956(1999)). This
may be partially due to the inclusion of the disulfide-linked
dimerization domain, which improves stability of scAbm on the
surface.
[0263] Virus-cell binding assay was employed to examine whether
psc.alpha.CD20 retained its binding activity. The supernatant of
sc.alpha.CD20-bearing lentiviruses (designated as
FUGW/sc.alpha.CD20+SINmu) was incubated with 293T/CD20 cells and
the resulting virus-cell complexes were analyzed by FACS. It was
found that FUGW/sc.alpha.CD20+SINmu viruses were able to bind to
CD20-expressing 293T cells, indicating that sc.alpha.CD20 on the
viral surface was active.
[0264] The ability of FUGW/sc.alpha.CD20+SINmu to specifically
transduce CD20.sup.+ cells was next investigated. Lentiviruses
carrying sc.alpha.CD20 can transduce 293T/CD20cells expressing
CD20. The titer was estimated to be around 5.times.10.sup.5 IU/mL.
Lentiviruses incorporating a single chain antibody had a somewhat
lower titer than those incorporating the natural form of antibody,
possibly because of the ability of the natural form of the antibody
to induce endocytosis. Nevertheless, these results demonstrated
that scAbm can be used to generate lentiviruses capable of
transducing cells expressing cognate receptors.
EXAMPLE 8
Specific Infection of Cells Expressing Surface-bound Anti-CD20
Antibodies Using an Engineered Recombinant Lentivirus Carrying CD20
and Fusion Proteins
[0265] Experiments to address (1) whether surface proteins other
than antibodies can be used to target lentiviral vectors, and (2)
whether surface receptors other than CD20 could be targeted for
cell-specific transduction were performed.
[0266] The use of CD20 to target cells expressing .alpha.CD20 was
investigated. Unlike CD20, which is a 4-transmembrane protein,
membrane-bound IgG.sub.1 has a C-terminal transmembrane and
cytoplasmic portions that anchor the molecule in the plasma
membrane. Physiologically the cytoplasmic domains can mediate
internalization of antigen-immunoglobulin complexes (Nussenzweig,
M. C. Curr. Biol. 7, R355-357(1997)). 293T cells were stably
generated expressing membrane-bound .alpha.CD20, designated herein
as 293T/.alpha.CD20. Harnessing the nature of the budding
mechanism, recombinant lentiviruses carrying CD20 and HAmu/SINmu
(designated as FUGW/CD20+HAmu and FUGW/CD20+SINmu, respectively)
were prepared. Infectivity of lentiviruses bearing CD20 was
measured by transducing 293T/.alpha.CD20 and quantifying GFP
expression. FUGW/CD20+HAmu virus can specifically transduce 293T
cells expressing .alpha.CD20. The titer was estimated to be about
1.2.times.10.sup.6 IU/mL. This data indicates that the
membrane-bound antibody can act as a viral receptor to mediate
entry of lentiviruses carrying the cognate antigen.
[0267] Therefore, it was demonstrated that transmembrane proteins
such as CD20 can be incorporated into the viral surface to target
the lentiviral vectors, expanding the pool of proteins that can be
exploited for targeting strategies. These results also show to one
of ordinary skill in the art that many different types of cell
surface receptors can be utilized as affinity molecules to mediate
the targeted entry.
EXAMPLE 9
Specific Infection of Cells Expressing c-kit Using an Engineered
Recombinant Lentivirus Co-displaying the Membrane-bound Form of
Stem Cell Factor (SCF) and Fusion Proteins
[0268] It was investigated whether other surface receptor-ligand
interactions could be exploited to achieve targeting. Stem cell
factor (SCF) interacts with c-kit, a protein tyrosine kinase
receptor on cell surfaces, to modulate hemopoiesis. (Shimizu, Y. J.
et al. J. Immunol. 156, 3443-34491996)). It was found that
engagement of SCF with c-kit led to rapid internalization of c-kit
via the endosomal pathway (Jahn, T. et al. Oncogene 21,
4508-4520(2002)). Casimir et al. showed that membrane-bound human
SCF could be incorporated into ecotropic retroviruses and found
that the resulting viruses were able to specifically transduce
c-kit-expressing human cells (Chandrashekran, A., et al. Blood 104,
2697-2703(2004)). Original ecotropic viruses do not infect human
cells because the envelope protein Eco cannot recognize mCAT
expressed on the human cell surface (the mCAT derived from rodent
cells is the viral receptor for Eco; Coffin, J. M. et al. (1997).
Retroviruses (New York, Cold Spring Harbor Laboratory Press)). The
ability of HAmu and SINmu to coordinate with SCF to target
recombinant lentiviruses and retroviruses was evaluated.
[0269] The membrane-bound form of human SCF (designated as hSCF)
was cloned from the s1/s14 hSCF220 stromal cell line (obtained from
ATCC). To construct the membrane-bound form of mouse SCF
(designated as mSCF), the cDNA for secreted mouse SCF was linked
with the transmembrane portion of hSCF. It was found that both hSCF
and mSCF could be stably displayed on the surface of lentiviruses.
When HAmu and hSCF were engineered into recombinant lentiviruses
(FUGW/hSCF+HAmu), the viruses, failed to infect TF-1a cells
expressing human c-kit. Similarly, FUGW/mSCF+HAmu could not infect
D9 cells expressing mouse SCF receptor. On the other hand, when
SINmu was used, FUGW/mSCF+SINmu and FUGW/mSCF+SINmu were able to
specifically infect c-kit positive cells TF-1a and D9 respectively.
Their titers were estimated to be about 1.5.times.10.sup.6 IU/mL
and 4.times.10.sup.6 IU/ml, respectively. These results indicate
the appropriate coordination between fusion and recognition protein
is an important factor for the recombinant virus to be
infectious.
[0270] When retroviruses displaying mSCF and SINmu were displayed
to infect D9 cells, extremely high infectivity (93%) was obtained.
Retroviruses carrying only mSCF or SINmu exhibited virtually no
infection of D9 cells. The infectivity was as high as what Eco
pseudotyped viruses could achieve. This indicates that receptor
ligand pairs can be used to target retroviruses to particular
target cells.
* * * * *