U.S. patent application number 12/154439 was filed with the patent office on 2008-12-18 for methods and compositions for increased transgene expression.
This patent application is currently assigned to Sangamo BioSciences, Inc.. Invention is credited to Michael C. Holmes, Gary Ka Leong Lee.
Application Number | 20080311095 12/154439 |
Document ID | / |
Family ID | 39760956 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080311095 |
Kind Code |
A1 |
Holmes; Michael C. ; et
al. |
December 18, 2008 |
Methods and compositions for increased transgene expression
Abstract
Described herein are methods of expressing nucleic acids in T
cells pre-exposed to a co-stimulatory signal and then transduced
with adenoviral vectors. In some embodiments, the co-stimulation is
provided by anti-CD3 and anti-CD28 antibodies and the adenoviral
vector is pseudotyped for T-cell entry. The invention also relates
to compositions for carrying out these methods, provided as kits or
pharmaceutical compositions that can be used to treat diseases
including immunological conditions and hematological
malignancies.
Inventors: |
Holmes; Michael C.;
(Oakland, CA) ; Lee; Gary Ka Leong; (San Leandro,
CA) |
Correspondence
Address: |
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD, SUITE 230
PALO ALTO
CA
94303
US
|
Assignee: |
Sangamo BioSciences, Inc.
|
Family ID: |
39760956 |
Appl. No.: |
12/154439 |
Filed: |
May 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60939825 |
May 23, 2007 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
435/326 |
Current CPC
Class: |
A61P 25/00 20180101;
C12N 5/0636 20130101; A61P 31/16 20180101; A61P 19/10 20180101;
C12N 15/86 20130101; A61P 37/04 20180101; C12N 2510/02 20130101;
A61P 1/02 20180101; C12N 2501/51 20130101; A61P 33/02 20180101;
A61P 33/06 20180101; A61P 27/02 20180101; C12N 2501/2315 20130101;
A61P 1/04 20180101; A61P 31/22 20180101; A61P 7/04 20180101; A61P
13/12 20180101; A61P 19/02 20180101; A61P 31/10 20180101; A61P 7/06
20180101; A61P 31/06 20180101; A61P 29/00 20180101; A61P 31/14
20180101; C12N 15/63 20130101; A61P 31/20 20180101; A61P 21/04
20180101; C12N 2501/515 20130101; A61P 1/16 20180101; A61P 11/06
20180101; A61P 17/02 20180101; A61P 31/18 20180101; C12N 2710/10343
20130101; A61P 35/00 20180101; A61P 37/02 20180101; A61P 37/06
20180101; A61P 7/00 20180101; A61P 3/10 20180101; A61P 17/00
20180101; A61P 33/04 20180101; A61P 35/02 20180101; A61P 31/08
20180101; A61P 31/04 20180101 |
Class at
Publication: |
424/93.21 ;
435/326 |
International
Class: |
A61K 45/00 20060101
A61K045/00; C12N 5/16 20060101 C12N005/16; A61P 37/02 20060101
A61P037/02 |
Claims
1. A method for increasing expression of an exogenous sequence in T
cells, said method comprising: a) activating a population of T
cells with at least a first and second co-stimulatory agents; and
b) contacting the activated T cell population with an adenoviral
expression vector comprising said exogenous sequence; wherein the
contacting results in expression of the exogenous sequence in
greater than 50% of said activated T cells.
2. The method according to claim 1, wherein the T cell is a CD4+
cell.
3. The method according to claim 1, wherein the T cell is a CD8+
cell.
4. The method according to claim 1, wherein at least one of the
co-stimulatory agents comprises an anti-CD3 antibody.
5. The method according to claim 4, wherein the second
co-stimulatory agent comprises an antiCD28 antibody.
6. The method according to claim 5, wherein the antibodies are
provided on a bead.
7. The method according to claim 4, wherein the second
co-stimulatory agent comprises IL-15.
8. The method according to claim 4, wherein the second
co-stimulatory agent comprises a feeder cell.
9. The method according to claim 1, wherein the adenoviral
expression vector is pseudotyped.
10. The method according to claim 9, wherein the pseudotyped
adenovirus expression vector comprises sequences from Ad5 and Ad35
adenoviruses.
11. The method according to claim 10, wherein the Ad35 sequence is
F35.
12. The method according to claim 9, wherein the pseudotyped
adenovirus expression vector comprises sequences from Ad5 and Ad 11
adenoviruses.
13. The method according to claim 1 wherein said adenoviral
expression vector further comprises a sequence encoding at least
one zinc finger nuclease.
14. The method according to claim 13, wherein the zinc finger
nuclease binds to a target site in CCR5.
15. A kit for expressing an exogenous sequence in T cells
comprising at least one co-stimulatory agent and an adenoviral
vector.
16. A method for cleaving an endogenous gene, the method comprising
expressing at least one zinc finger nuclease in a T cell according
to the method of claim 1, such that the zinc finger nuclease
cleaves the endogenous gene.
17. The method of claim 16, wherein the endogenous gene is a CCR5
gene.
18. The method of claim 16, wherein the endogenous gene is a GR
gene.
19. A pharmaceutical composition comprising at least one
co-stimulatory agent and an adenovirus vector comprising an
exogenous sequence.
20. A pharmaceutical composition comprising: T-cells obtained by
activation and transduction of T cells with a pharmaceutical
composition according to claim 19 and a pharmaceutically acceptable
carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/939,825, filed May 23, 2007, the
disclosure of which is hereby incorporated by reference in its
entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
TECHNICAL FIELD
[0003] The present invention relates generally to improved methods
and compositions for expressing exogenous nucleic acids in cells,
and more particularly to improved expression of exogenous genetic
material in human immune cells using adenoviral vectors.
BACKGROUND
[0004] Reliable transgene expression in immune effector cells such
as T cells can provide needed insight into immunological
mechanisms, and would also help enable novel immunotherapeutic
strategies such as, e.g., gene therapy approaches for treating
cancer as well as immunological, hematological, infectious, and
genetic disorders. Unfortunately, however, stable and effective
transgene expression in T cells has proven to bean elusive goal,
often requiring hit-or-miss experimentation. It is
well-established, for example, that transduction of resting primary
T lymphocytes with viral vectors, such as retroviral derived
vectors, generally results in very low efficiencies [Yamashita and
Emerman, J. Virology 2006 Jan. 5; 344(1):88-93]. Accordingly,
studies in retroviral-mediated gene transfer to primary T
lymphocytes over the past ten years have examined a number of
different strategies to improve transduction efficiencies, leaving
the even greater hurdle of satisfactory transgene expression as a
more distant goal.
[0005] With respect to viral systems providing permanent
integration into the host cell, it has been shown that T cell
activation in conjunction with lentiviral transduction can
significantly improve gene delivery to T cells. See, e.g., Unutmaz
et al., J Exp Med. 1999 Jun. 7; 189(11):1735-46; Pollock et al., J.
Virology, 1998 June; 72(6):4882-92; Costello et al., Gene Ther.
2000 April; 7(7):596-604; Bai et al., Gene Ther. 2003 August;
10(17):1446-57; Lu et al., J Gene Med. 2004 September; 6(9):963-73.
Bai et. al. and Lu et. al., for example, independently reported
that both phytohemagluttinin (PHA) and anti-CD3/antiCD28
costimulation of T lymphocytes can improve lentiviral gene delivery
efficiencies, demonstrating 80 to >99% transduction. Despite
these reported successes in transduction efficiency, however, the
actual levels of gene expression obtained by these researchers is
inconclusive based on the reported data.
[0006] Unfortunately, however, attempts to reproduce these results
using non-integrative viral systems have been more problematic.
See, e.g., Schroers et al., Exp Hematol. 2004 June; 32(6):53646. In
the Schroers study, PHA activation provided only a modest
improvement in transduction efficiency with adenoviral vectors,
approaching only 45 percent transduction, significantly lower than
that obtained using lentiviral vectors. Thus, there remains a
significant need in the art for improved methods and compositions
capable of high transgene expression in T cells in non-integrative
viral systems, e.g., to provide proteins of therapeutic,
industrial, or research uses. The present invention addresses this
and other needs.
SUMMARY
[0007] The present invention derives from the surprising finding
that a T cell co-stimulatory signal, unlike other forms of T cell
activation, significantly improves adenoviral-mediated transduction
of T cells with exogenous nucleic acids and, more importantly,
produces high expression levels of an encoded exogenous molecule.
The compositions and methods described herein allow for increased
transient expression of any exogenous sequence in any T cell.
Accordingly, the need for an integrating vector (retrovirus or
lentivirus), which vectors can cause insertional mutagenesis or
variegated expression due to position effects, is eliminated. In
addition, the ability to transiently express exogenous sequences in
T cells may provide for more stable cell populations, for example
in that immunogenic peptides may be cleared out before the host
immune system can mount a response. Thus, as described herein, the
exogenous molecule may be any diagnostic or a therapeutic molecule
of interest, thus providing novel methods of diagnosing, treating
and/or preventing a wide variety of conditions and disorders.
[0008] In one aspect, methods for increasing expression of an
exogenous nucleic acid in T cells are provided, comprising 1)
activating a population of T cells in vitro or ex vivo with a
co-stimulatory signal produced by at least a first and second
co-stimulatory agent; and 2) contacting said activated T cell
population with an adenoviral expression vector comprising said
exogenous nucleic acid; wherein said contacting results in
expression of a transgene encoded by the exogenous nucleic acid in
greater than 50% of said activated T cells. In one embodiment, the
T cell is a CD4+ T cell. In another embodiment, the T cell is a
CD8+ T cell.
[0009] In a preferred embodiment, the co-stimulatory signal
includes activation of the T cell receptor, and employs a
co-stimulatory agent comprising a CD3 ligand. In a particularly
preferred embodiment, the CD3 ligand comprises an anti-CD3
antibody. In certain embodiments, expression of the endogenous gene
is at least about three fold greater, preferably at least about
five fold greater and even more preferably at least about six fold
greater as compared to T cell activated with PHA and/or IL 2 and
transduced with an adenoviral vector.
[0010] In a preferred embodiment, the co-stimulatory signal also
includes activation of the CD28 receptor, and the co-stimulatory
agent further comprises a CD28 ligand. In a particularly preferred
embodiment the CD28 ligand comprises a CD28 antibody.
[0011] In an alternative embodiment, the co-stimulatory agent
further comprises IL-15. In another alternative embodiment, the
co-stimulatory signal further comprises a feeder cell.
[0012] In a preferred embodiment, the adenoviral expression vector
is pseudotyped to enhance targeting of immune cells, e.g., T cells.
In one embodiment, the pseudotyped adenovirus expression vector
comprises sequences from Ad5 and Ad35 adenoviruses. In a particular
embodiment, the Ad35 sequence is F35. In another embodiment, the
pseudotyped adenovirus expression vector comprises sequences from
Ad5 and Ad 11 adenoviruses.
[0013] In one aspect, the exogenous nucleic acid encodes a
diagnostic molecule such as, e.g. a fluorescent protein (e.g., GFP
or RFP).
[0014] In another aspect, the exogenous nucleic acid encodes a
therapeutic molecule such as, e.g., an RNA molecule or a
therapeutic protein. In preferred embodiments, the therapeutic
protein is selected from the group consisting of hormones, enzymes,
cytokines, chemokines, antibodies, mitogenic factors, growth
factors, differentiation factors, factors influencing angiogenesis,
factors influencing cell apoptosis, and factors influencing
inflammation.
[0015] In another aspect, the exogenous nucleic acid encodes for a
molecule capable of disrupting, enhancing or altering endogenous
gene expression such as, e.g., transcription factors or nucleases.
In preferred embodiments, the exogenous nucleic acid encodes for a
zinc finger protein. In a particular embodiment, the zinc finger
protein is directed to CCR5.
[0016] In another aspect, the exogenous nucleic acid sequence may
produce one or more non-coding sequences, for example, one or more
RNA molecules (e.g., small hairpin RNAs (shRNAs), small interfering
RNAs (siRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs),
etc.).
[0017] In one aspect, the invention provides an improved method of
integrating an exogenous sequence into a T cell, said method
comprising: a) activating said T cell with a co-stimulatory signal;
and exposing said activated T cell to an adenoviral vector
comprising said exogenous sequence, for example, using ZFN-mediated
targeted integration. In certain embodiments, the integration of
the exogenous sequence is at least about 70% to 90% efficient,
including any value therebetween (e.g., 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%).
[0018] In one aspect, kits for expressing and/or integrating an
exogenous sequence in T cells are provided comprising a
costimulatory agent as described herein and an adenoviral
expression vector.
[0019] In one aspect, methods of cleaving an endogenous gene in a
cell are provided comprising expressing at least one zinc finger
nuclease in a T cell according to any of the methods described
herein, such that the zinc finger nuclease(s) cleave(s) the
endogenous gene. In certain embodiments, the zinc finger nuclease
cleaves a gene encoding a receptor involved in HIV entry into a
cell (e.g., CCR5). In other embodiments, the zinc finger
nuclease(s) cleave(s) a glucocorticoid receptor (GR). Thus, also
provided are methods of preventing and/or treating HIV by
expressing a CCR-5 binding zinc finger nuclease in a T cell.
Methods of retaining T cell immune function during glucocorticoid
treatment by cleaving a GR are also provided.
[0020] Additional features and advantages of the invention will be
set forth in the detailed description which follows, and in part
will be readily apparent to those skilled in the art from that
description or recognized by practicing the invention as described
herein, including the detailed description-which follows, the
claims, as well as the appended figures.
[0021] It is to be understood that both the foregoing general
description and the following detailed description present
embodiments of the invention, and are intended to provide an
overview or framework for understanding the nature and character of
the invention as it is claimed. The accompanying drawings are
included to provide a further understanding of the invention, and
are incorporated into and constitute a part of this specification.
The figures illustrate various embodiments of the invention, and
together with the description serve to explain the principles and
operations of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 illustrates transduction and expression of GFP in
primary T lymphocytes using either Ad5 or Ad5/F35 GFP adenoviral
vectors, following activation with anti-CD3/anti-CD28
antibodies.
[0023] FIG. 2, panels A and B, are graphs illustrating transduction
and expression levels of GFP in CD4+ cells using either Ad5 or
Ad5/F35 GFP adenoviral vectors, following activation with IL-7
(FIG. 2A); or (B) anti-CD3/anti-CD28 antibodies (FIG. 2B).
[0024] FIG. 3, panels A and B, are graphs illustrating transduction
and expression using 2 different lots of Ad5/F35 GFP adenoviral
vectors, following activation with anti-CD3/anti-CD28 antibodies,
in either primary T lymphocytes (FIG. 3A); or CD4+ cells (FIG.
3B).
[0025] FIG. 4 is a graph illustrating transduction obtained in CD4+
cells over time, following activation with either PHA or
anti-CD3/anti-CD28 antibodies at MOIs of 30 or 100.
[0026] FIG. 5, panels A and B, are graphs depicting transduction
(FIG. 5A) and expression (FIG. 5B) of GFP Day 1 after transduction
in CD4+ cells using Ad5/F35 GFP adenoviral vectors, following
activation with either PHA/IL 2 or antiCD3/anti-CD28
antibodies.
[0027] FIG. 6, panels A and B, are graphs depicting transduction
(FIG. 6A) and expression (FIG. 6B) of GFP Day 2 after transduction
in CD4+ cells using AdSlF35 GFP adenoviral vectors, following
activation with either PHA/IL 2 or antiCD3/anti-CD28
antibodies.
[0028] FIG. 7 is a graph depicting GFP expression obtained in CD4+
cells over time, following activation with either PHA or
anti-CD3/anti-CD28 antibodies each the indicated MOIs of either 30
or 100.
[0029] FIG. 8 illustrates T-cell expansion over time, following
activation with either PHA or antiCD3/anti-CD28 antibodies each at
MOIs of 0, 10, 30 or 100.
[0030] FIG. 9, panels A and B, show gels depicting differences in
CCR5 gene modification in CD4+ T cells exposed to Ad5/F35 vectors
carrying a zinc finger nuclease directed to CCR5 when
pre-stimulated with either PHA or anti-CD3/anti-CD28 antibodies.
Lane 1 shows non-transduced cells; lane 2 shows cells transduced
with an Ad5/F35 encoding GFP, lane 3 shows cells transduced with
ZFNs targeting the IL2Ry gene, lane 4 shows cells transduced with
ZFNs targeting CCR5, and lane 5 shows cells transduced with a
second set of CCR5ZFNs. CCR5 and IL2R.gamma.-targeted ZFNs are
described, for example, in International Patent Publication Nos. WO
2007/139982; 2005/014791 and WO 2005/084190. At the indicted days
post-transduction, an aliquot of cells were harvested and genomic
DNA harvested using the MasterPure.TM. DNA Purification Kit
(Epicentre Biotechnologies). The level of ZFN activity in response
to different activation conditions was assessed by measuring the
efficiency of gene modification at the endogenous CCR5 locus using
the Surveyor assay. The percentage of NHEJ events is shown beneath
each lane.
DETAILED DESCRIPTION
[0031] Reference will now be made in detail to the present
preferred embodiments of the invention, examples of which are
illustrated in the accompanying figures.
[0032] The present invention relates to methods and compositions
for transducing exogenous nucleic acids into T cells using
non-integrative viral vectors such as, e.g., adenoviral vectors,
and particularly to the expression of transgenes contained therein.
The invention is at least partly based on the surprising results
obtained with respect to high transgene expression as well as
transduction efficiency in T cells when activated with a
co-stimulatory signal and transduced with adenoviral vectors
carrying the exogenous genetic material.
[0033] One aspect of the present invention relates to methods for
expressing exogenous nucleic acids in T cells. In preferred
embodiments, the invention provides improved methods for
introducing exogenous nucleic acids encoding a transgene into a
cell, where the transgene is expressed. An "exogenous nucleic acid"
can be defined as any nucleic acid introduced into a cell. The
exogenous nucleic acid may encode a polypeptide product.
Alternatively, the exogenous nucleic acid sequence may produce one
or more non-coding sequences, for example, one or more RNA
molecules (e.g., small interfering RNAs (siRNAs), small hairpin
RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs), etc.).
The exogenous nucleic acid may or may not correspond to a sequence
(e.g., gene) otherwise found or expressed in the cell. For example,
the "exogenous" nucleic acid may correspond to a gene normally
found in healthy T cells, but not found or found to a lesser
extent, or expressed to a lesser extent, in the T cells being
transduced. "Transgene" as used herein refers to an exogenous gene
that is introduced into cells to be expressed, i.e., transcribed
and/or translated into a polypeptide product. "Exogenous nucleic
acid" is also used interchangeably herein with "exogenous genetic
material."
[0034] The term "nucleic acid" as used herein can refer to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form. The term is used to indicate, for example,
genes, cDNAs, and mRNAs. For the purposes of the present
disclosure, these terms are not to be construed as limiting with
respect to the length of a polymer. The terms can encompass known
analogues of natural nucleotides, as well as nucleotides that are
modified in the base, sugar and/or phosphate moieties. In general,
an analogue of a particular nucleotide has the same base-pairing
specificity; i.e., an analogue of A will base-pair with T. The term
also encompasses nucleic acids containing modified backbone
residues or linkages, which are synthetic, naturally occurring, and
non-naturally occurring. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-0-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
[0035] The T cells transduced by the methods of the present
invention can be any T cell of any mammalian animal. As used
herein, "T cell" is used interchangeably with "T lymphocyte." In
preferred embodiments, the T cell is a primary T lymphocyte, more
preferably a human primary T lymphocyte. In some preferred
embodiments, the T cell is a CD4+ cell. The T cell can also be a
CD8+ cell, or a mixture of CD4+ and CD8+ cells. T cells for use in
the subject compositions and methods can be obtained and/or
accessed by methods well known in the art. Samples can be used
immediately or frozen for future use. Frozen samples can be thawed
and cultured in appropriate media before use, according to methods
known in the art.
Co-Stimulatory Signals
[0036] The methods of the present invention involve activating the
T cells with a co-stimulatory signal. A "co-stimulatory signal" as
used herein refers to a coordinated activation signal employing at
least two co-stimulatory agents. In preferred embodiments, the
co-stimulatory signal includes activation of the T cell receptor,
and the first co-stimulatory agent comprises a CD3 ligand, and
preferably an anti-CD3 antibody. In preferred embodiments for T
cells, and CD4+ T cells in particular, the co-stimulatory signal
further includes activation of the CD28 receptor, and the second
costimulatory agent comprises a CD28 ligand, and preferably an
anti-CD28 antibody. In alternative embodiments for CD8+ T cells,
the co-stimulatory signal further includes activation of the IL-15
receptor, and the second co-stimulatory agent comprises IL-15. In
some embodiments, the antibodies are provided on beads such as,
e.g., Dynalbead CD3/CD28 (Invitrogen, Carlsbad, Calif.).
[0037] While co-stimulation of the CD3 and CD28 pathways are
required for optimal transduction of CD4+ T cells, as shown herein,
additional hematopoietic cell lineages also require co-stimulation
through at least two distinct pathways (e.g. a mitogen signal and a
signal associated with cell-cell contact) to achieve complete
activation. Accordingly, the methods described herein may be
applicable to other cell types, using similar adenoviral vectors
but co-stimulatory signals appropriate for the particular cell
type. Examples include, but are not limited to, CD8+ T cells
co-stimulated by anti-CD3 and CD 137ligand (4-1BBL) [Wen et al., J.
Immunol. 2002 May 15; 168(10):4897-906).], B lymphocytes
co-stimulated by CD40L and IL4 [Fecteau et al.; J Immunol. 2003
Nov. 1; 171(9):4621-9], and subsets of NK cells being co-stimulated
by anti-CD94 antibodies and IL2 or IL15 [Voss et at., J. Immunol.
1998 Feb. 15; 160(4):1618-26.].
Expression Vectors
[0038] A further step of the present methods involves exposing the
activated cells to a vector carrying exogenous genetic material to
be expressed. The term "vector" as used herein refers to a carrier
nucleic acid molecule into which a nucleic acid sequence can be
inserted for introduction into a cell where, in some embodiments,
it can be replicated. In preferred embodiments, the vector is an
expression vector. By "expression vector" is meant a vector
containing the appropriate control sequences to permit
transcription and, optionally, translation of an exogenous gene in
a transduced cell. Typically, the expression vector includes an
"expression cassette," which comprises a nucleic acid to be
transcribed operably linked to a promoter. For example, the
expression vector may contain a promoter sequence in the regulatory
region which facilitates the transcription of (and is operably
linked to) an inserted nucleic acid sequence. An expression vector
also includes a vector that directs transcription of a sequence,
for example, one or more RNA molecules (e.g., small hairpin RNAs
(shRNAs), small interfering RNAs (siRNAs), inhibitory RNAs (RNAis),
microRNAs (miRNAs), etc.)
[0039] In more preferred embodiments, the vector is an adenoviral
expression vector. Adenoviruses contain a linear double-stranded
DNA molecule of approximately 36,000 base pairs with identical
inverted terminal repeats ("ITRs") of approximately 90-140 base
pairs. The exact length of the ITRs depends on the adenovirus
serotype. Adenoviruses have been used as vectors to achieve
transduction and transgene expression in various cell types by
capitalizing on their mechanisms of infection. For example, using
Ad5 vectors, the initial step for successful infection is binding
of adenovirus to its target cell, a process mediated through their
fiber proteins. The fiber protein has a trimeric structure
(Stouten, P. W. F., Sander, C., Ruigrok, R. W. H., and Cusack, S.
(1992) New triple helical model for the shaft of the adenovirus
fiber. J. Mol. Biol. 226, 1073-1084) with different lengths
depending on the virus serotype (Signas, G., Akusjarvi, G., and
Petterson, U. (1985). Adenovirus 3 fiberpolypeptide gene:
Complications for the structure of the fiber protein. Journal of
Virology. 53, 672-678; Kidd, A. H., Chrboczek, J., Cusack, S., and
Ruigrok, R. W./H. (1993) Adenovirus type 40 virions contain two
distinct fibers. Virology 192, 73-84). Different serotypes have
polypeptides with structurally similar N and C termini, but
different middle stem regions. The first 30 amino acids at the N
terminus are involved in anchoring of the fiber to the penton base
(Chroboczek J., Ruigrok R. W. H., and Cusack S., 1995. Adenovirus
fiber, p. 163-200. In: W. Doerfer and P. Bohm (ed.), The molecular
repertoire of adenoviruses, I. Springer-Verlag, Berlin), especially
the conserved FNPVYP region in the tail (Amberg N., Mei Y. and
Wadell G., 1997. Fiber genes of adenoviruses with tropism for the
eye and the genital tract. Virology 227: 239-244). The C-terminus,
or "knob", is responsible for initial interaction with the cellular
adenovirus receptor. After this initial binding, secondary binding
between the capsid penton base and cell-surface integrins leads to
internalization of viral particles in coated pits and endocytosis
(Morgan, C., Rozenkrantz, H. S., and Mednis, B. (1969) Structure
and development of viruses as observed in the electron
microscope.X. Entry and uncoating of adenovirus. J. Virol 4,
777-796; Svensson, V. and Persson, R. (1984). Entry of adenovirus 2
into Hela cells. Journal of Virology. 51, 687-694; Varga, M. J.,
Weibull, C., and Everitt, E. (1991). Infectious entry pathway of
adenovirus type 2. Journal of Virology 65, 6061-6070; Greber, U.
F., Willets, M., Webster, P., and Helenius, A. (1993). Stepwise
dismanteling of adenovirus 2 during entry into cells. Cell 75,
477-486; Wickham, T. J., Mathias, P., Cherish, D. A., and Nemerow,
G. R. (1993) Integrins avb3 and avb5 promote adenovirus
internalisation but not virus attachment. Cell 73, 309-319).
Integrins are .alpha..beta.-heterodimers of which at least 14
.alpha.-subunits and 8 .beta.-subunits have been identified (Hynes,
R. O. (1992) Integrins: versatility, modulation and signaling in
cell adhesion. Cell 69, 11-25). The array of integrins expressed in
cells is complex and will vary between cell types and cellular
environment. Although the knob contains some conserved regions,
between serotypes, knob proteins show a high degree of variability,
indicating that different adenovirus receptors exist.
[0040] Some other adenoviral vectors may use other mechanisms for
entry. For example, subgroup B adenoviruses, such as Ad35, utilize
human CD46 as a cellular attachment receptor (Gaggar et. al. (2003)
Nature Medicine, Vol 9, pp. 1408-1412). It has also been
demonstrated that chimeric Ad5135 vectors possess the same receptor
specificity as Ad35, thus allowing for CAR-independent transduction
of cells, such as human hematopoetic cells (Nilsson et. al.,
(2004), Molecular Therapy, Vol. 9, pp. 377-388).
[0041] At least six different subgroups of human adenoviruses have
been proposed, encompassing approximately 50 distinct adenovirus
serotypes. Besides these human adenoviruses, many animal
adenoviruses have been identified (see, e.g., Ishibashi, M. and
Yasue (1983) in Adenoviruses of Animals, Chapter 12, p497-561). A
serotype can be defined on the basis of its immunological
distinctiveness as determined by quantitative neutralization with
animal antiserum (horse, rabbit). If neutralization shows a certain
degree of cross-reaction between two viruses, distinctiveness of
serotype is assumed if A) the hemagglutinins are unrelated, e.g.,
as shown by lack of cross-reaction on hemagglutination-inhibition,
or B) substantial biophysical/biochemical differences in DNA exist
(Francki, R. I. B., Fauquet, C. M., Knudson, D. L. and Brown, F.
(1991) Classification and nomenclature of viruses. Fifth report of
the international Committee on taxonomy of viruses. Arch. Virol.
Suppl. 2, 140-144). Besides differences towards the sensitivity
against neutralizing antibodies of different adenovirus serotypes,
adenoviruses in subgroup C such as Ad2 and Ad5 bind to different
receptors as compared to adenoviruses from subgroup B such as Ad3,
Ad7, Ad11, Ad14, Ad21, Ad34, and Ad35 (see, e.g., Defer C., Belin
M., Caillet-Boudin M. and Boulanger P., 1990. Human adenovirus-host
cell interactions; comparative study with members of subgroup B and
C.
[0042] Journal of Virology 64 (8): 3661-3673; Gall J., Kass-Eisler
A., Leinwand L. and Falck-Pedersen E., 1996. Adenovirus type 5 and
7 capsid chimera: fiber replacement alters receptor tropism without
affecting primary immune neutralization epitopes. Journal of
Virology 70 (4): 2116-2123).
[0043] Pseudotyping of adenoviral vectors can be made possible
primarily by swapping the knob region of the fiber protein of one
adenovirus serotype with that of another serotype [Stevenson, J.
Virol. 1997 June; 71(6):4782-90]. This type of fiber protein
manipulation allows for modification of the vector tropism, i.e.,
allows for transduction of a different range of cell types
[Mizuguchi 2002 (Adenovirus vectors containing chimeric type 5 and
type 35 fiber proteins exhibit altered and expanded tropism and
increase the size limit of foreign genes. February 20;
285(1-2):69-77., Takayama et al., Virology. 2003 May 10;
309(2):282-93.); Stecher et al. Mol. Ther. 2001 July; 4(1):36-44].
Construction of such chimeric adenoviral vectors is known in the
art. For example, it has been demonstrated that receptor
specificity could be altered by exchanging the Ad3 knob protein
with the Ad5 knob protein, and vice versa (Krasnykh V. N., Mikheeva
G. V., Douglas J. T. and Curiel D. T., 1996. Generation of
recombinant adenovirus vectors with modified fibers for altering
viral tropism. Journal of Virology 70(10): 6839-6846; Stevenson S.
C., Rollence M., White B., Weaver L. and McClelland A., 1995. Human
adenovirus serotypes 3 and 5 bind to two different cellular
receptors via the fiber head domain. Journal of Virology 69(5):
2850-2857 Stevenson S. C., Rollence M., Marshall-Neff J. and
McClelland A., 1997. Selective targeting of human cells by a
chimeric adenovirus vector containing a modified fiber protein.
Journal of Virology 71(6): 4782-4790; see also, e.g., Yotnda et
al., Gene Ther. 2001 June; 8(12):930-7).
[0044] In some embodiments, the adenovirus expression vector is
pseudotyped. For example, the vector can comprise a chimeric
adenoviral vector, that is, a genetically modified vector
comprising nucleic acid sequences from two or more adenoviruses
(plus an exogenous gene). Typically, the nucleic acid sequences
will correspond to adenoviral proteins from adenoviruses of two
different serotypes. In preferred embodiments, the vector comprises
a sequence corresponding to at least one tissue tropism determining
fragment of a fiber protein derived from a subgroup B adenovirus,
where the tropism is directed towards T-cells. In more preferred
embodiments, the vector comprises sequences from Ad5 and Ad35
adenoviruses, for example, where the Ad5 fiber genes are
substituted by the Ad35 fiber genes. In still more preferred
embodiments, the Ad35 sequences corresponding to the Ad35 gene
encode the knob portion of the fiber protein, known as F35. Such a
vector can be referred to as an Ad5/F35 vector. In some
embodiments, the vector comprises Ad5 and Ad11 sequences. In still
some other embodiments, a combination of adenoviral vectors can be
used, e.g., a combination of any of the kinds of adenoviral
expression vectors and/or chimeric adenoviral vectors described
herein.
[0045] Those of skill in the art will recognize other modifications
or alterations that can be made to the adenoviral vector in the
practice of some embodiments of the instant invention. For example,
in some embodiments, the adenoviral vector is modified such that
the capacity of the adenoviral nucleic acid to replicate in a
target cell is reduced or disabled. In some embodiments, the
adenoviral nucleic acid is modified so that the capacity of a host
immune system to mount an immune response against adenoviral
proteins encoded by the adenovirus nucleic acid is reduced or
disabled. In some embodiments, the adenoviral nucleic acid is
modified so as to be integrative into the host cell genome. For
example, in some embodiments homologous sequences are used to
integrate at least a portion of adenoviral nucleic acid into the
host cell's genome, e.g., via targeted homologous
recombination.
[0046] According to methods of the instant invention, the vector is
allowed to transduce the T-cell, e.g., by exposing the T-cells to
the vector under conditions suitable for transduction. For example,
T lymphocyte cultures can be incubated at about 37.degree. C., 5%
CO.sub.2. A vector can be said to "transduce" a cell where the
vector enters the cell. Entry may be by any process, e.g.,
receptor-mediated endocytosis, by which particles are taken up by
cell. As outlined in more detail above, uptake of some adenovirus
particles is generally a two-stage process involving an initial
interaction of the fiber protein of the virus with cellular
receptors, such as the MHC class 1 molecule and the
coxsackievirus-adenovirus receptor. Viral penton proteins then bind
to integrin cell receptors, facilitating internalization via
receptor-mediated endocytosis. For example, this is the mechanism
used by A5 vectors. Other adenoviral vectors, e.g., AD5/35 vectors,
utilize CD46 for entry. Adenovirus vectors can be readily
constructed using methods known in the art and/or commercially
available kits such as the AdEasy.TM. Kits (Strategene, La Jolla,
Calif.).
[0047] In still some embodiments of the instant invention, one or
more of the adenoviral vectors described herein are used in
combination with other approaches known in the art. For example, a
mixture of adenoviral and lentiviral vectors may be used to achieve
transduction.
[0048] As known to those in the art, vectors can be added directly
to the T cells at various dilutions to achieve varying MOIs
(multiplicities of infection). For example, vectors can be diluted
with culture medium an MOI in the range of about 10 to about 1000
infectious units per cell. One advantage obtained from use of the
present invention is that the MOI can be reduced to levels that are
less toxic to human immune cells and T cells in particular, while
still achieving efficient adenovirus transduction and an adequate
level of transgene expression.
[0049] In some preferred embodiments, a CD4+ cell is activated
using beads coated with anti-CD3 and anti-CD28 antibodies and
transduced with Ad5/F35 vectors carrying the exogenous gene to be
introduced.
[0050] Following transduction, the exogenous gene can then be
expressed in the T cells. As described in greater detail in the
Examples below, some embodiments of the instant invention provided
surprisingly and unexpectedly higher transgene expression levels
compared to that obtained using methods described previously (e.g.,
using lentiviral gene delivery). Moreover, methods of some
embodiments of the instant invention produce at least about 3-fold,
at least about 5-fold, at least about 10-fold, at least about
15-fold or at least about 20-fold improvement over using no T cell
activation.
[0051] For example, in preferred embodiments, the methods provided
herein result in increased levels of expression of the exogenous
gene as compared with methods previously described in the art. For
example, methods herein can provide at least about 2-fold, at least
about 3-fold, or at least about 4-fold the level of expression
obtained using no co-stimulatory signal. Moreover, the present
invention can also provide improved levels of expression compared
with known methods for activating the cells. For example, compared
to pre-stimulation with PHA or IL 2, methods of some embodiments of
the instant invention provide at least about 3-fold, at least about
4-fold, at least about 5-fold or at least about 6-fold the level of
transgene expression See, e.g., Examples provided below where
transgene expression levels are measured via mean fluorescent
intensity of GFP, using a GFP gene as the transgene.
[0052] In some embodiments, methods of the instant invention
provide at least about 3-fold, at least abut 5-fold, at least about
10-fold or even at least about 15-fold the level of transgene
expression when compared to pre-stimulation with PHA or IL2. See,
e.g., Examples provided below where transgene expression levels are
measured via the rate of NHEJ events caused by expression of a
nuclease encoded as the transgene. Also see FIG. 11, that
illustrates the difference in CCR5 gene modification in CD4+ T
cells exposed to Ad5/F35 vectors carrying a zinc finger nuclease
directed to CCR5 when pre-stimulated with either PHA or
anti-CD3/anti-CD28 antibodies.
[0053] Transduction can result in either transient or stable
expression. Where the transgene becomes stably integrated into a
host's genome, for example, stable expression can be obtained. In
some applications, transient expression is more preferred. For
example, one approach involves using almost homologous sequences
that differ from a region on the host's genome by one nucleotide.
Integration in such cases can occur at high frequencies, e.g., 1 in
10 or 1 in 20, but as cells divide, the nucleotide difference is
corrected. This approach can achieve high transduction efficiencies
and high but transient transgene expression.
[0054] One of skill in the art will also recognize applications
where the exogenous nucleic acid introduced into a cell need not be
expressed. For example, methods of some embodiments of the present
invention can deliver nucleic acid sequences that are useful in
themselves. Nucleic acid sequences that are transcribed but not
translated include, for example, aptamers, ribosomal RNA, tRNA,
splicosomal RNA, antisense RNA, siRNA, shRNA, miRNA and mRNA.
Accordingly, another aspect of the instant invention relates to
methods for transducing T-cells by activating the T-cell with a
costimulatory signal, exposing it to an adenoviral vector carrying
the exogenous gene and allowing the vector to enter the T-cell. The
cells, vectors and co-stimulatory signals described above can also
be used with respect to this aspect of the invention. Preferred
embodiments of the instant invention provide for increased
transduction efficiencies with respect to introducing exogenous
genetic material into T cells. For example, in some embodiments,
transduction is at least about 60%, at least abut 65%, at least
about 70% or, at least about 80%, efficient. Nucleic acid delivery
efficiencies can be measured by various techniques known in the
art. Examples include, but are not limited to, flow cytometry, Cel
I assay, and the like.
Kits.
[0055] Another aspect of the instant invention involves kits for
transducing T-cells and/or for expressing exogenous genes in
T-cells. Such kits can be constructed by packaging the appropriate
materials, including one or more compositions of the instant
invention, preferably with appropriate labels, in suitable
containers. Kits can also include additional reagents and materials
(for example, suitable buffers, salt solutions, etc.) helpful for
carrying out the procedures and/or for measuring the degree of
transduction and/or transgene expression. In some embodiment, the
kits also include a suitable set of instructions pertaining to the
transduction and/or transgene expression methods disclosed
herein.
[0056] In some embodiments, the kit comprises a co-stimulatory
agent and an adenovirus vector, e.g., and adenoviral expression
vector. A "co-stimulatory agent" as used herein can refer to any
compound, composition, aggregate, solution, etc. that can provide a
co-stimulatory signal for activation of a T cell. Examples include,
but are not limited to, beads coated with anti-CD3 antibodies,
beads coated with anti-CD3 and anti-CD28 antibodies, and IL-15, in
combinations as described herein.
[0057] The adenovirus vector of the kit can be one or more of any
of the adenoviral vectors described herein. In preferred
embodiments, the vector is an adenoviral expression vector
pseudotyped to facilitate transduction of T cells. In a specific
embodiment, the vector comprises an Ad5/F35 vector. In some
embodiments, the vector also carries an exogenous gene for
introduction and/or expression in T cells. In other embodiments,
the vector is provided without the foreign insert, e.g., where the
vector is provided with adenoviral sequences and instruction for
inserting an exogenous gene of choice by the user.
[0058] Those of skill in the art will recognize various uses for
the improved methods and compositions described herein. For
example, methods of the instant invention can be used in ex vivo
applications, where higher transduction efficiency (in terms of
percent transduced) and/or higher transgene expression levels are
desired. Such applications include, but are not limited to gene
modification, characterization of gene function, and altering
properties of T-cells.
Gene Modification
[0059] With respect to gene modification, enhanced delivery and/or
expression of nucleases can lead to increased gene disruption, gene
alteration, and/or targeted integration. For example, in such
applications the exogenous genetic material can comprise a nuclease
such as zinc finger nucleases (ZFN), discussed in more detail
below, such as CCR5-ZFNs (8267 and 8196z) and the GR-ZFNs (9674 and
9666). Integration may occur via NHEJ and/or through homologous
directed repair donor (HDR), events to introduce the exogenous gene
into a region altering a targeted gene and/or randomly disrupting
various genes. HDR events involve providing a donor molecule with
sequence homologous to the ZFN target site that can be used as the
template for the HDR machinery.
[0060] In some embodiments, the vector used for transduction
comprises a zinc finger pair. A zinc finger pair refers to two
binding domains of one or more zinc finger proteins. The term "zinc
finger protein" or "ZFP" refers to a protein having DNA binding
domains that are stabilized by zinc. The individual DNA binding
domains are typically referred to as "fingers." A ZFP has least one
finger, typically two fingers, three fingers, four fingers, five
fingers or six fingers. Each finger binds from two to four base
pairs of DNA, typically three or four base pairs of DNA. A ZFP
binds to a nucleic acid sequence called a target site or target
segment. Each finger typically comprises an approximately 30 amino
acid, zinc-chelating, DNA-binding subdomain. An exemplary motif
characterizing one class of these proteins (C.sub.2H.sub.2 class)
is -Cys-(X).sub.2-4-Cys-(X).sub.12-His-(X).sub.3-5-His (where X is
any amino acid) (SEQ ID NO:29). Studies have demonstrated that a
single zinc finger of this class consists of an alpha helix
containing the two invariant histidine residues co-ordinated with
zinc along with the two cysteine residues of a single beta turn
(see, e.g., Berg & Shi, Science 271:1081-1085 (1996)).
Non-canonical (i.e., non-C2H2) zinc finger proteins are also
described, for example in U.S. Pat. Nos. 7,273,923 and 7,262,054
and U.S., Patent Publication Nos. 20060246588; 20060246567; and
20030108880.
[0061] Zinc finger binding domains can be engineered to bind to a
predetermined nucleotide sequence. See, e.g., U.S. Application
Publication No. 2007/0059795. In preferred embodiments, e.g., the
zinc finger pair can be directed to CCR5. In more preferred
embodiments, e.g., the vector carries both a zinc finger pair
directed to CCR5 and a nuclease. (a ZFN), whereby the nuclease can
specifically cut the CCR5 gene, as elaborated below.
[0062] Alternatively, DNA-binding domain may be derived from a
nuclease. For example, the recognition sequences of homing
endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI,
PI-Sce, I-SceIV, I-CsmI, I-PanI; I-SceII, I-PpoI, I-SceIII, I-Crel,
I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. No.
5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic
Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118;
Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996)
Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.
263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the
New England Biolabs catalogue. In addition, the DNA-binding
specificity of homing endonucleases and meganucleases can be
engineered to bind non-natural target sites. See, for example,
Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al.
(2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006)
Nature 441:656-659; Paques et al. (2007) Current Gene Therapy
7:49-66; U.S. Patent Publication No. 20070117128.
Zinc Finger Nucleases
[0063] Described herein are zinc finger nucleases (ZFNS) that can
be used for gene inactivation, for example inactivation of the CCR5
gene. ZFNs comprise a zinc finger protein (ZFP) domain and a
nuclease (cleavage) domain. Exemplary disclosures of zinc finger
nucleases, and methods for their synthesis and use, are provided in
U.S. Patent Application Publication Nos. 2003/0232410;
2005/0026157; 2005/0064474; 2005/0208489; and 2006/0188987; and PCT
Publications WO 2005/84190 and WO 2007114275. Exemplary
CCR-5-targeted ZFNs, and methods for their synthesis and use, are
disclosed in International Patent Publication WO 2007/139982, also
incorporated in entirety herein.
A. Zinc Finger Proteins
[0064] Zinc finger binding domains can be engineered to bind to a
sequence of choice. See, for example, Beerli et al. (20.02) Nature
Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.
70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660;
Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al.
(2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc
finger binding domain can have a novel binding specificity,
compared to a naturally-occurring zinc finger protein. Engineering
methods include, but are not limited to, rational design and
various types of selection. Rational design includes, for example,
using databases comprising triplet (or quadruplet) nucleotide
sequences and individual zinc finger amino acid sequences, in which
each triplet or quadruplet nucleotide sequence is associated with
one or more amino acid sequences of zinc fingers which bind the
particular triplet or quadruplet sequence. See, for example,
co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261 and U.S. Patent
Application Publication No. 2004/0197892, all of which are
incorporated by reference herein in their entireties. Design
methods are also disclosed in U.S. Pat. Nos. 6,013,453; 6,479,626;
6,746,838; 6,866,997; 6,903,185; 7,030,215 and 7,153,949; and WO
01/53480, the disclosures of which are incorporated by reference in
their entireties.
[0065] Exemplary selection methods, including phage display and
two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538;
5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759;
6,242,568; 6,790,941; as well as WO 98/37186; WO 98/53057; WO
00/27878; WO 01/88197 and GB 2,338,237; the disclosures of which
are incorporated by reference in their entireties. Enhancement of
binding specificity for zinc finger binding domains has been
described, for example, in co-owned U.S. Pat. No. 6,794,136.
[0066] In certain embodiments, the zinc finger domain of the zinc
finger nucleases described herein binds to a target sequence in a
CCR-5 gene. Table 1 describes a number of zinc finger binding
domains that have been engineered to bind to nucleotide sequences
in the human CCR-5 gene. Each row describes a separate zinc finger
DNA-binding domain. The DNA target sequence for each domain is
shown in the first column (DNA target sites indicated in uppercase
letters; non-contacted nucleotides indicated in lowercase), and the
second through fifth columns show the amino acid sequence of the
recognition region (amino acids -1 through +6, with respect to the
start of the helix) of each of the zinc fingers (F1 through F4) in
the protein. Also provided in the first column is an identification
number for each protein.
TABLE-US-00001 TABLE 1 Zinc finger nucleases targeted to the human
CCR-5 gene Target sequence F1 F2 F3 F4 r162 designs GATGAGGATGAC
DRSNLSR (SEQ TSANLSR (SEQ RSDNLAR (SEQ TSANLSR (SEQ (SEQ ID NO:1)
7296 ID NO:2) ID NO:3) ID NO:4) ID NO:3) GATGAGGATGAC DRSNLSR (SEQ
ISSNLNS (SEQ RSDNLAR (SEQ TSANLSR (SEQ (SEQ ID NO:1) 8181 ID NO:2)
ID NO:5) ID NO:4) ID NO:3) GATGAGGATGAC DRSNLSR (SEQ VSSNLTS (SEQ
RSDNLAR (SEQ TSANLSR (SEQ (SEQ ID NO:1) 8182 ID NO:2) ID NO:6) ID
NO:4) ID NO:3) GATGAGGATGAC DRSNLSR (SEQ ISSNLNS (SEQ RSDNLAR (SEQ
NRDNLSR (SEQ (SEQ ID NO:1) 8262 ID NO:2) ID NO:5) ID NO:4) ID NO:7)
GATGAGGATGAC DRSNLSR (SEQ ISSNLNS (SEQ RSDNLAR (SEQ TSGNLTR (SEQ
(SEQ ID NO:1) 8266 ID NO:2) ID NO:5) ID NO:4) ID NO:8) GATGAGGATGAC
DRSNLSR (SEQ VSSNLTS (SEQ RSDNLAR (SEQ TSGNLTR (SEQ (SEQ ID NO:1)
8267 ID NO:2) ID NO:6) ID NO:4) ID NO:8) GATGAGGATGAC DRSNLSR (SEQ
TSGNLTR (SEQ RSDNLAR (SEQ TSGNLTR (SEQ (SEQ ID NO:1) 87741 ID NO:2)
ID NO:8) ID NO:4) ID NO:8) 168 designs AAACTGCAAAAG RSDNLSV (SEQ
QNANRIT (SEQ RSDVLSE (SEQ QRNHRTT (SEQ (SEQ ID NO:9) 7745 ID NO:10)
ID NO:11) ID NO:12) ID NO:13) AAACTGCAAAAG RSDNLSN (SEQ QNANRIT
(SEQ RSDVLSE (SEQ QRNHRTT (SEQ (SEQ ID NO:9) 8165 ID NO:14) ID
NO:11) ID NO:12) ID NO:13) AAACTGCAAAAG RSDNLSV (SEQ QRVNLIV (SEQ
RSDVLSE (SEQ QRNHRTT (SEQ (SEQ ID NO:9) 8191 ID NO:10) ID NO:15) ID
NO:12) ID NO:13) AAACTGCAAAAG RSDNLGV (SEQ QKINLQV (SEQ RSDVLSE
(SEQ QRNHRTT (SEQ (SEQ ID NO:9) 8196 ID NO:16) ID NO:17) ID NO:12)
ID NO:13) AAACTGCAAAAG RSDNLSV (SEQ QKINLQV (SEQ RSDVLSE (SEQ
QRNHRTT (SEQ (SEQ ID NO:9) 8196z ID NO:10) ID NO:17) ID NO:12) ID
NO:13) AAACTGCAAAAG RSDNLGV (SEQ QKINLQV (SEQ RSDVLSE (SEQ QRNHRTT
(SEQ (SEQ ID NO:9) ID NO:16) ID NO:17) ID NO:12) ID NO:13) 8196zg
AAACTGCAAAAG RSDHLSE (SEQ QNANRIT (SEQ RSDVLSE (SEQ QRNHRTT (SEQ
(SEQ ID NO:9) 7568 ID NO:18) ID NO:11) ID NO:12) ID NO:13) r627
designs GACAAGCAGCGG RSAHLSE (SEQ RSANLSE (SEQ RSANLSV (SEQ DRANLSR
(SEQ (SEQ ID NO:19) 7524 ID NO:20) ID NO:21) ID NO:22) ID NO:23 633
designs CATCTGcTACTCG RSDSLSK (SEQ DNSNRIK (SEQ RSAVLSE (SEQ
TNSNRIT (SEQ (SEQ ID NO:24) 8040 ID NO:25) ID NO:26) ID NO:27) ID
NO:28)
[0067] In certain embodiments, a zinc finger binding domain as
shown in Table I is fused to a cleavage half-domain, such as, for
example, the cleavage domain of a Type IIs restriction endonuclease
such as Fokl. A pair of such zinc finger/nuclease half-domain
fusions are used for targeted cleavage, as disclosed, for example,
in U.S. Patent Application Publication No. 2005/0064474. For
example, ZFN-215 denotes the pair of fusion proteins containing the
zinc finger binding domains designated 8267 (which recognizes the
target sequence shown in SEQ ID NO:1 and comprises the 4
recognition helices depicted in SEQ ID NOs:2, 6, 4 and 8) and 8196z
(which recognizes the target sequence shown in SEQ ID NO:9 and
comprises the 4 recognition helices depicted in SEQ ID NOs:10, 17,
12 and 13). ZFN-201 denotes the pair of fusion proteins containing
the zinc finger binding domains designated 8266 (which recognizes
the target sequence shown in SEQ ID NO:1 and comprises the 4
recognition helices depicted in SEQ ID NOs: 2, 2, 4 and 8) and
8196z (which recognizes the target sequence shown in SEQ ID NO:9
and comprises the 4 recognition helices depicted in SEQ ID NOs:10,
17, 12 and 13).
[0068] For targeted cleavage, the near edges of the binding sites
can separated by 5 or more nucleotide pairs, and each of the fusion
proteins can bind to an opposite strand of the DNA target. Hence,
any one of the proteins identified as an "r162 design" in Table 1
(indicating that it binds to the reverse strand and that the
downstream edge of its binding site is at nucleotide 162) can be
paired with any of the proteins identified as a "168 design`
(indicating that it binds to the strand opposite that bound by the
r162 designs and that the upstream edge of its binding site is at
nucleotide 168). For example, protein 8267 can be paired with
protein 8196 or with protein 8196z or with any of the other 168
designs; and protein 8266 can be paired with either of proteins
8196 or 8196z or with any other of the 168 designs. All pairwise
combinations of the r162 and 168 designs can be used for targeted
cleavage and mutagenesis of a CCR-5 gene. Similarly, the 7524
protein (or any other r627 design) can be used in conjunction with
the 8040 protein (or any other 633 design) to obtain targeted
cleavage and mutagenesis of a CCR-5 gene.
[0069] The CCR5-ZFNs described herein can be targeted to any
sequence in the CCR5 genome. For example, CCR5 genomic sequences
(including allelic variants such as CCR5-A32) are well known in the
art and individuals homozygous for the CCR5-432 (see, e.g., Liu et
al. (1996) Cell 367377), are resistant to HIV-1 infection.
B. Cleavage Domains
[0070] The cleavage domain portion of the fusion proteins disclosed
herein can be obtained from any endonuclease or exonuclease.
Exemplary endonucleases from which a cleavage domain can be derived
include, but are not limited to, restriction endonucleases and
homing endonucleases. See, for example, 2002-2003 Catalogue, New
England Biolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic
Acids Res. 25:3379-3388. Additional enzymes which cleave DNA are
known (e.g., S1 Nuclease; mung bean nuclease; pancreatic DNase I;
micrococcal nuclease; yeast HO endonuclease; see also Linn et al.
(eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). Non
limiting examples of homing endonucleases and meganucleases include
I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceUV, I-CsmI, I-PanI, I-SceII,
I-PpoI, I-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII are known.
See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort
et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al (1989)
Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,
1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al.
(1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol.
Biol. 280:345-353 and the New England Biolabs catalogue. One or
more of these enzymes (or functional fragments thereof) can be used
as a source of cleavage domains and cleavage half-domains.
[0071] Similarly, a cleavage half-domain can be derived from any
nuclease or portion thereof, as set forth above, that requires
dimerization for cleavage activity. In general, two fusion proteins
are required for cleavage if the fusion proteins comprise cleavage
half-domains. Alternatively, a single protein comprising two
cleavage half-domains can be used. The two cleavage half-domains
can be derived from the same endonuclease (or functional fragments
thereof), or each cleavage half-domain can be derived from a
different endonuclease (or functional fragments thereof). In
addition, the target sites for the two fusion proteins are
preferably disposed, with respect to each other, such that binding
of the two fusion proteins to their respective target sites places
the cleavage half-domains in a spatial orientation to each other
that allows the cleavage half-domains to form a functional cleavage
domain, e.g., by dimerizing. Thus, in certain embodiments, the near
edges of the target sites are separated by 5-8 nucleotides or by
15-18 nucleotides. However any integral number of nucleotides or
nucleotide pairs can intervene between two target sites (e.g., from
2 to 50 nucleotide pairs or more). In general, the site of cleavage
lies between the target sites.
[0072] Restriction endonucleases (restriction enzymes) are present
in many species and are capable of sequence-specific binding to DNA
(at a recognition site), and cleaving DNA at or near the site of
binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at
sites removed from the recognition site and have separable binding
and cleavage domains. For example, the Type IIS, enzyme Fok I
catalyzes double-stranded cleavage of DNA, at 9 nucleotides from
its recognition site on one strand and 13 nucleotides from its
recognition site on the other. See, for example, U.S. Pat. Nos.
5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992)
Proc. Natl. Acad. Sci. USA 89:42754279; Li et al. (1993) Proc.
Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Nat'l.
Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.
269:31,978-31,982. Thus, in one embodiment, fusion proteins
comprise the cleavage domain (or cleavage half-domain) from at
least one Type IIS restriction enzyme and one or more zinc finger
binding domains, which may or may not be engineered.
[0073] An exemplary Type IIS restriction enzyme, whose cleavage
domain is separable from the binding domain, is Fok I. This
particular enzyme is active as a dimer. Bitinaite et al. (1998)
Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Accordingly, for the
purposes of the present disclosure, the portion of the Fok I enzyme
used in the disclosed fusion proteins is considered a cleavage
half-domain. Thus, for targeted double-stranded cleavage and/or
targeted replacement of cellular sequences using zinc finger-Fok I
fusions, two fusion proteins, each comprising a Fok1 cleavage
half-domain, can be used to reconstitute a catalytically active
cleavage domain. Alternatively, a single polypeptide molecule
containing a zinc finger binding domain and two Fok I cleavage
half-domains can also be used. Parameters for targeted cleavage and
targeted sequence alteration using zinc finger-Fok I fusions are
provided elsewhere in this disclosure.
[0074] A cleavage domain or cleavage half-domain can be any portion
of a protein that retains cleavage activity, or that retains the
ability to multimerize (e.g., dimerize) to form a functional
cleavage domain.
[0075] Exemplary Type IIS restriction enzymes are described in U.S.
Patent Application Publication No. 2005/0064474 and International
Patent Publication WO 2007/014275, incorporated herein in their
entireties. Additional restriction enzymes also contain separable
binding and cleavage domains, and these are contemplated by the
present disclosure. See, for example, Roberts et al. (2003) Nucleic
Acids Res. 31:418-420.
[0076] To enhance cleavage specificity, cleavage domains may also
be modified. In certain embodiments, variants of the cleavage
half-domain are employed, which variants that minimize or prevent
homodimerization of the cleavage half-domains. Non-limiting
examples of such modified cleavage half-domains are described in
detail in WO 2007/014275, incorporated by reference in its entirety
herein. In certain embodiments, the cleavage domain comprises an
engineered cleavage half-domain (also referred to as dimerization
domain mutants) that minimize or prevent homodimerization are known
to those of skill the art and described for example in U.S. Patent
Publication Nos. 20050064474 and 20060188987, incorporated by
reference in their entireties herein. Amino acid residues at
positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498,
499, 500, 531, 534, 537, and 538 of Fok I are all targets for
influencing dimerization of the Fok I cleavage half-domains. See,
e.g., U.S. Patent Publication Nos. 20050064474 and 20060188987;
International Patent Publication WO 07/139,898; Miller et al.
(2007) Nat. Biotechnol. 25(7):778-785.
[0077] Exemplary engineered cleavage half-domains of Fok I that
form obligate heterodimers include a pair in which a first cleavage
half-domain includes mutations at amino acid residues at positions
490 and 538 of Fok I and a second cleavage half-domain includes
mutations at amino acid residues 486 and 499. See FIGS. 2, 3 and 4
of International Patent Publication WO 2007/139982, incorporated
herein by reference.
[0078] Thus, in one embodiment, as shown in FIGS. 3 and 4, the
mutation at 490 replaces Glu (E) with Lys (K); the mutation at 538
replaces Iso (I) with Lys (K); the mutation at 486 replaced Gin (Q)
with Glu (E); and the mutation at position 499 replaces Iso (I)
with Lys (K). Specifically, the engineered cleavage half-domains
described herein were prepared by mutating positions 490 (E to K)
and 538 (I to K) in one cleavage half-domain to produce an
engineered cleavage half-domain designated "E490K: 1538K" and by
mutating positions 486 (Q to E) and 499 (I to L) in another
cleavage half-domain to produce an engineered cleavage half-domain
designated "Q486E: 1499L". The engineered cleavage half-domains
described herein are obligate heterodimer mutants in which aberrant
cleavage is minimized or abolished. See, e.g., Example 1 of
International Patent Publication No. 07/139,898, the disclosure of
which is incorporated by reference in its entirety for all
purposes.
[0079] Engineered cleavage half-domains described herein can be
prepared using any suitable method, for example, by site-directed
mutagenesis of wild-type cleavage half-domains (Fok I) as described
in U.S. Patent Publication No. 2005/0064474 (e.g., Example 5) and
WO 2007/14275 (e.g., Example 38).
C. Additional Methods for Targeted Cleavage in CCR5
[0080] Any nuclease having a target site in a CCR5 gene can be used
in the methods disclosed herein. For example, homing endonucleases
and meganucleases have very long recognition sequences, some of
which are likely to be present, on a statistical basis, once in a
human-sized genome. Any such nuclease having a unique target site
in a CCR5 gene can be used instead of, or in addition to, a zinc
finger nuclease, for targeted cleavage in a CCR5 gene.
[0081] Exemplary homing endonucleases include I-Scel, I-Ceul,
PI-Pspl, PI-Sce, I-SceIV, I-Csml, IPanl, I-Scell, I-Ppol, I-Scell,
I-Crel, I-TevI, I-Tevll and I-TevIII. Their recognition sequences
are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No.
6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388;
Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic
Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;
Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al.
(1998) J. Mol. Biol. 280:345-353 and the New England Biolabs
catalogue.
[0082] Although the cleavage specificity of most homing
endonucleases is not absolute with respect to their recognition
sites, the sites are of sufficient length that a single cleavage
event per mammalian sized genome can be obtained by expressing a
homing endonuclease in a cell containing a single copy of its
recognition site; It has also been reported that the specificity of
homing endonucleases and meganucleases can be engineered to bind
non-natural target sites. See, for example, Chevalier et al. (2002)
Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.
31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et
al. (2007) Current Gene Therapy 7:49-66.
Gene Characterization
[0083] With respect to characterizing gene function, improved
delivery and/or expression of cDNA cassettes can up-regulate to a
greater extent expression of a gene being studied. As another
example, improved delivery and/or expression of siRNA expression
cassettes can down-regulate to a greater extent expression of a
gene being studied. In either case, the enhanced change in
expression can allow better characterization of gene function of
T-cell genes.
[0084] Ex vivo applications also include altering the functional
properties of a pool of transduced T cells. Examples of transgenes
that can be used to alter T-cell function include, but are not
limited to, chimeric T-cell receptors that can re-target a pool of
T-cells against a specific antigen; and cytokines that can be
over-expressed to enhance immune function and/or anti-viral
activity, as provided in more detail below.
Treatments
[0085] The methods and compositions described herein can also find
use in the treatment of a number of hematological, immunological
and/or genetic conditions in animal subjects. The term "treating"
(and its grammatical variants) as used herein includes achieving a
therapeutic benefit and/or a prophylactic benefit. By therapeutic
benefit is meant eradication or amelioration of the underlying
condition being treated. Also, a therapeutic benefit is achieved
with the eradication or amelioration of one or more of the
physiological symptoms associated with the underlying condition
such that an improvement is observed in the subject,
notwithstanding the fact that the subject may still be afflicted
with the condition. For prophylactic benefit, a composition of the
present invention may be administered to a subject at risk of
developing a condition, or to a subject reporting one or more of
the physiological symptoms of such a condition, even though a
diagnosis may not have been made.
[0086] The invention also provides pharmaceutical compositions for
administration to a subject that can be treated using one or more
of the methods and/or compositions disclosed herein. In some
embodiments, pharmaceutical compositions comprise a therapeutic
gene as the exogenous gene carried by the adenoviral vector and
introduced into host cells ex vivo. By "therapeutic gene" is meant
a nucleic acid molecule that when transduced and/or expressed
produces a beneficial result in a subject receiving treatment.
Examples of such therapeutic genes include IL2, which enhances
T-cell expansion and can find use in treatment of patients with
cancer; as well as CD40L (gp39 or CD154), which plays a role in TIC
cell interaction and APC maturation and can find use in treatment
of patients with certain types of leukemia. As a specific example,
it has been demonstrated that an allosterically controllable
ribozyme induces cell death in chronic myelogenous leukemia (CML)
cell line harboring the b2a2 type bcr-abl oncogene in vitro and in
vivo. Tanabe et al., Nature 2000, 406: 473-474. Some embodiments of
the instant invention can find use in transducing the ribozyme into
primary T cells of patients with CML, e.g., where the ribozyme can
be transduced and expressed at high levels using one or more
approaches taught herein.
Immunological Disorders
[0087] In one embodiment, the therapeutic proteins expressed by the
transduced T cells possess immunomodulatory activity. For example,
a therapeutic polypeptide of the present invention may be useful in
treating deficiencies or disorders of the immune system, by
activating or inhibiting the proliferation, differentiation, or
mobilization (chemotaxis) of immune cells. Immune cells develop
through the process of hematopoiesis, producing myeloid (platelets,
red blood cells, neutrophils, and macrophages) and lymphoid (B and
T lymphocytes) cells from pluripotent stem cells. The etiology of
these immune deficiencies or disorders may be genetic, somatic,
such as cancer or some autoimmune disorders, acquired (e.g. by
chemotherapy or toxins), or infectious.
[0088] A therapeutic polypeptide of the present invention may be
useful in treating deficiencies or disorders of hematopoietic
cells. A therapeutic polypeptide of the present invention could be
used to increase differentiation or proliferation of hematopoietic
cells, including the pluripotent stem cells, in an effort to treat
those disorders associated with a decrease in certain (or many)
types hematopoietic cells. Examples of immunologic deficiency
syndromes include, but are not limited to: blood protein disorders
(e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia
telangiectasia, common variable immunodeficiency, Digeorge
Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion
deficiency syndrome, lymphopenia, phagocyte bactericidal
dysfunction, severe combined immunodeficiency (SCIDs),
Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or
hemoglobinuria.
[0089] A therapeutic polypeptide of the present invention may also
be useful in treating autoimmune disorders. Many autoimmune
disorders result from inappropriate recognition of self as foreign
material by immune cells. This inappropriate recognition results in
an immune response leading to the destruction of the host tissue.
Therefore, the administration of a therapeutic polypeptide of the
present invention that inhibits an immune response, particularly
the proliferation, differentiation, or chemotaxis of T-cells, may
be an effective therapy in preventing autoimmune disorders.
[0090] Examples of autoimmune disorders that can be treated by the
present invention include, but are not limited to: Addison's
Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid
arthritis, dermatitis, allergic encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,
Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura,
Reiter's Disease, Stiff Man Syndrome, Autoimmune Thyroiditis,
Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin-dependent diabetes mellitis,
Crohn's disease, ulcerative colitis, and autoimmune inflammatory
eye disease.
[0091] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by a therapeutic polypeptide of the present
invention.
[0092] Moreover, these molecules can be used to treat anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[0093] A therapeutic polypeptide of the present invention may also
be used to treat and/or prevent organ rejection or
graft-versus-host disease (GVHD). Organ rejection occurs by host
immune cell destruction of the transplanted tissue through an
immune response. Similarly, an immune response is also involved in
GVHD, but, in this case, the foreign transplanted immune cells
destroy the host tissues. The administration of a therapeutic
polypeptide of the present invention that inhibits an immune
response, particularly the proliferation, differentiation, or
chemotaxis of T-cells, may be an effective therapy in preventing
organ rejection or GVHD.
[0094] Similarly, a therapeutic polypeptide of the present
invention may also be used to modulate inflammation. For example,
the therapeutic polypeptide may inhibit the proliferation and
differentiation of cells involved in an inflammatory response.
These molecules can be used to treat inflammatory conditions, both
chronic and acute conditions, including inflammation associated
with infection (e.g. septic shock, sepsis, or systemic inflammatory
response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated hyperacute rejection,
nephritis, cytokine or chemokine induced lung injury, inflammatory
bowel disease, Crohn's disease, or resulting from over production
of cytokines (e.g. TNF or IL-1).
[0095] Examples of classes of proteins having immunomodulatory
activity include cytokines and thymic hormones. Thymic hormones
include, for example, prothymosin-.alpha., thymulin, thymic humoral
factor (THF), THF-.gamma.-2, thymocyte growth peptide (TGP),
thymopoietin (TPO), thymopentin, and thymosin-.alpha.-1. The term
cytokine refers to a diverse group of secreted, soluble proteins
and peptides that mediate communication among cells and modulate
the functional activities of individual cells and tissues. Classes
of cytokines include interleukins, interferons, colony stimulating
factors, and chemokines. Examples of cytokines include:
IL-1.alpha., IL-1.beta., IL-2 through IL-30, leukocyte inhibitory
factor (LIF), IFN-.alpha., IFN-.gamma., TNF, TNF-.alpha.,
TGF-.beta., G-CSF, M-CSF, and GM-CSF. One or more cytokines can be
introduced and over-expressed in T cells, e.g., to enhance immune
function and/or anti-viral properties.
Hyperproliferative Disorders
[0096] In one embodiment, a therapeutic protein of the invention is
capable of modulating cell proliferation. Such a therapeutic
polypeptide can be used to treat hyperproliferative disorders,
including neoplasms.
[0097] Examples of hyperproliferative disorders that can be treated
by a therapeutic polypeptide of the present invention include, but
are not limited to neoplasms located in the: abdomen, bone, breast,
digestive system, liver, pancreas, peritoneum, endocrine glands
(adrenal, parathyroid, pituitary, testicles, ovary, thymus,
thyroid), eye, head and neck, nervous (central and peripheral),
lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and
urogenital.
[0098] Similarly, other hyperproliferative disorders can also be
treated by a therapeutic polypeptide of the present invention.
Examples of such hyperproliferative disorders include, but are not
limited to: hypergammaglobulinemia, lymphoproliferative disorders,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,
Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis,
and any other hyperproliferative disease, besides neoplasia,
located in an organ system listed above.
[0099] A therapeutic polypeptide expressed by a transgene according
to the present invention may inhibit the proliferation of the
disorder through direct or indirect interactions. Systemic
administration of the transduced cells provides for access of
therapeutic protein to a wide variety of tissues. Alternatively, a
therapeutic polypeptide of the present invention may stimulate the
proliferation of other cells which can inhibit the
hyperproliferative disorder.
[0100] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. This immune response
may be increased by either enhancing an existing immune response,
or by initiating a new immune response. Alternatively, decreasing
an immune response may also be a method of treating
hyperproliferative disorders, such as with a chemotherapeutic
agent.
Infectious Disease
[0101] In one embodiment, a therapeutic polypeptide of the present
invention can be used to treat infectious disease. For example, by
increasing the immune response, particularly increasing the
proliferation and differentiation of B and/or T cells, infectious
diseases may be treated. The immune response may be increased by
either enhancing an existing immune response, or by initiating a
new immune response. Alternatively, the therapeutic polypeptide of
the present invention may also directly inhibit the infectious
agent, without necessarily eliciting an immune response.
[0102] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated by a therapeutic
polypeptide of the present invention. Examples of viruses, include,
but are not limited to the following DNA and RNA viral families:
Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,
Flaviviridae, Hepadnaviridae (Hepatitis), HIV, Herpesviridae (such
as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus
(e.g. Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae (e.g. Influenza), Papovaviridae, Parvoviridae,
Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),
Reoviridae (e.g. Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g. Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
encephalitis, eye infections (e.g. conjunctivitis, keratitis),
chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active,
Delta), meningitis, opportunistic infections (e.g. AIDS),
pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever,
Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio,
leukemia, Rubella, sexually transmitted diseases, skin diseases
(e.g. Kaposi's, warts), and viremia. A therapeutic polypeptide of
the present invention can be used to treat any of these symptoms or
diseases.
[0103] With respect to HIV infection, one approach involves using
adenoviral vectors carrying zinc finger nucleases targeted to
regions of the CCR5 gene as the transgene, as described in more
detail above. See also, e.g., International Patent Publication WO
2007/139982, also incorporated in entirety herein. According to
some embodiments of the instant invention, improved transduction
and transgene expression can be achieved, resulting in efficient
targeting and cleavage within the CCR5.
[0104] Bacterial or fungal agents that can cause disease or
symptoms and that can be treated or detected by a therapeutic
polypeptide of the present invention include, but are not limited
to, the following Gram-Negative and Gram-positive bacterial
families and fungi: Actinomycetales (e.g. Corynebacterium,
Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g.
Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,
Borrelia, Brucellosis, Candidiasis, Campylobacter,
Coccidioidomycosis, Cryptococcosis, Dermatocycoses,
Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,
Listeria, Mycoplasmatales, Neisseriaceae (e.g. Acinetobacter,
Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g.
Actinobacillus, Heamophilus, Pasteurella), Pseudomonas,
Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal. These
bacterial or fungal families can cause the following diseases or
symptoms, including, but not limited to: bacteremia, endocarditis,
eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis,
opportunistic infections (e.g. AIDS related infections),
paronychia, prosthesis-related infections, Reiter's Disease,
respiratory tract infections, such as Whooping Cough or Empyema,
sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid
Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis,
Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,
Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g. cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections. A therapeutic polypeptide of the
present invention can be used to treat any of these symptoms or
diseases.
[0105] Moreover, parasitic agents causing disease or symptoms that
can be treated by a therapeutic polypeptide of the present
invention include, but are not limited to, the following families:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and
Trichomonas. These parasites can cause a variety of diseases or
symptoms, including, but not limited to: Scabies, Trombiculiasis,
eye infections, intestinal disease (e.g. dysentery, giardiasis),
liver disease, lung disease, opportunistic infections (e.g. AIDS
related), Malaria, pregnancy complications, and toxoplasmosis. A
therapeutic polypeptide of the present invention can be used to
treat any of these symptoms or diseases.
Regeneration
[0106] A therapeutic polypeptide of the present invention can be
used to differentiate, proliferate, and attract cells, fostering to
the regeneration of tissues. (See, Science 276:59-87 (1997).) The
regeneration of tissues could be used to repair, replace, or
protect tissue damaged by congenital defects, trauma (wounds,
burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery,
including cosmetic plastic surgery, fibrosis, reperfusion injury,
or systemic cytokine damage.
[0107] Tissues that could be regenerated with the contribution of a
therapeutic protein of the invention include organs (e.g. pancreas,
liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac), vascular (including vascular endothelium),
nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and
ligament) tissue. Preferably, regeneration incurs a small amount of
scarring, or occurs without scarring. Regeneration also may include
angiogenesis.
[0108] Moreover, a therapeutic polypeptide of the present invention
may increase regeneration of tissues difficult to heal. For
example, increased tendon/ligament regeneration would quicken
recovery time after damage. A therapeutic polypeptide of the
present invention could also be used prophylactically in an effort
to avoid damage. Specific diseases that could be treated include
tendinitis, carpal tunnel syndrome, and other tendon or ligament
defects. A further example of tissue regeneration of non-healing
wounds includes pressure ulcers, ulcers associated with vascular
insufficiency, surgical, and traumatic wounds.
Chemotaxis
[0109] In one embodiment, a therapeutic polypeptide of the present
invention possesses a chemotaxis activity. A chemotaxic molecule
attracts or mobilizes cells (e.g. monocytes, fibroblasts,
neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells) to a particular site in the body, such as
inflammation, infection, or site of hyperproliferation. The
mobilized cells can then fight off and/or heal the particular
trauma or abnormality.
[0110] A therapeutic polypeptide of the present invention may
increase the chemotaxic activity of the transduced cells. The
expressed chemotactic molecules can then be used to treat
inflammation, infection, hyperproliferative disorders, or any
immune system disorder by increasing the number of cells targeted
to a particular location in the body. For example, chemotaxic
molecules can be used to treat wounds and other trauma to tissues
by attracting additional immune cells to the injured location.
Chemotactic molecules of the present invention can also attract
fibroblasts, which can be used to treat wounds.
[0111] It is also contemplated that a therapeutic polypeptide of
the present invention may inhibit chemotactic activity. These
molecules could also be used to treat disorders. Thus, a
therapeutic polypeptide of the present invention could be used as
an inhibitor of chemotaxis.
[0112] Additional therapeutic polypeptides contemplated for use
include, but are not limited to, growth factors (e.g., growth
hormone, insulin-like growth factor-1, platelet-derived growth
factor, epidermal growth factor, acidic and basic fibroblast growth
factors, transforming growth factor-(3, etc.), to treat growth
disorders or wasting syndromes; and antibodies (e.g., human or
humanized), to provide passive immunization or protection of a
subject against foreign antigens or pathogens (e.g., H. Pylori), or
to provide treatment of cancer, arthritis or cardiovascular
disease; cytokines, interferons (e.g., interferon (INF), INF-a2b
and 2a, INF-aN1, INF-(31b, INF-gamma), interleukins (e.g., IL-1 to
IL 10), tumor necrosis factor (TNF-a TNF-R), chemokines,
granulocyte macrophage colony stimulating factor (GM-CSF),
polypeptide hormones, antimicrobial polypeptides (e.g.,
antibacterial, antifungal, antiviral, and/or antiparasitic
polypeptides), enzymes (e.g., adenosine deaminase), gonadotrophins,
chemotactins, lipid-binding proteins, filgastim (Neupogen),
hemoglobin, erythropoietin, insulinotropin, imiglucerase,
sarbramostim, tissue plasminogen activator (WA), urokinase,
streptokinase, phenylalanine ammonia lyase, brain-derived
neurotrophic factor (BDNF), nerve growth factor (NGF),
thrombopoietin (TPO), superoxide dismutase (SOD), adenosine
deamidase, catalase calcitonin, endothelian, L-asparaginase pepsin,
uricase trypsin, chymotrypsin elastase, carboxypeptidase lactase,
sucrase intrinsic factor, calcitonin parathyroid hormone
(PTH)-like, hormone, soluble CD4, and antibodies and/or
antigen-binding fragments (e.g., FAbs) thereof (e.g., orthoclone
OKT-3 (anti-CD3), GP11b/11a monoclonal antibody).
[0113] Those of skill in the art will recognize other therapeutic
gene products that can be expressed in transduced cells using
methods and/or compositions of various embodiments of the instant
invention. See, e.g., additional examples listed in US Application
Publication No. 20070059833. One of skill in the art will also
recognize the utility of methods disclosed herein to gene therapy.
In gene therapy, genetic information is usually delivered to a host
cell in order to correct (supplement) a genetic deficiency in the
cell, to inhibit an undesired function in the cell, or to eliminate
the host cell. The genetic information can also be intended to
provide the host cell with a desired function, for instance, to
supply a secreted protein to treat other cells of the host,
etc.
[0114] In some embodiments, one of more therapeutic gene products
may be purified for use in pharmaceutical preparations. In some
embodiments, the transgenic T cells themselves may be used
therapeutically, such that the pharmaceutical composition comprises
transgenic T cells, as described in more detail below.
[0115] In some embodiments, a pharmaceutically acceptable carrier
is included in the pharmaceutical composition. Pharmaceutically
acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions
available (see, e.g., Remington's Pharmaceutical Sciences, 17th
ed., 1989).
[0116] A pharmaceutical composition of the instant invention can be
administered to a subject in need thereof in an effective amount.
The effective amount when referring to a pharmaceutical composition
comprising T cells transduced as described herein, will generally
mean the dose ranges, modes of administration, formulations, etc.,
that have been recommended or approved by any of the various
regulatory or advisory organizations in the medical or
pharmaceutical arts (e.g., FDA, AMA) or by the manufacturer or
supplier. The effective amount when referring to producing a
benefit in treating a given condition, such as leukemia, will
generally mean the amount that achieves clinical results
recommended or approved by any of the various regulatory or
advisory organizations in the medical or surgical arts (e.g., FDA,
AMA) or by the manufacturer or supplier.
[0117] A person of ordinary skill using techniques known in the art
can determine the effective amount of the transduced T cell
described herein to be administered. The effective amount may
depend on the exogenous gene and/or co-stimulatory agent being
used, and can be deduced from known data, e.g., data regarding
desired expression of the exogenous gene, measures of the exogenous
gene obtained ex vivo (for example, as provided in the Examples
herein) and/or in animal models, along with knowledge of
translation of such data to the subject to be treated.
[0118] The pharmaceutical compositions can be administered by
various means known in the art. For example, vectors can be
delivered to cells ex vivo, such as cells explanted from an
individual patient (e.g., lymphocytes, bone marrow aspirates,
tissue biopsy), followed by re-implantation of the cells into a
patient, usually after selection for cells which have incorporated
the vector. Therapies where cells are genetically modified ex vivo,
and then re-introduced into a subject can be referred to as
cell-based therapies or cell therapies. In a preferred embodiment,
cells are isolated from the subject organism, transduced with an
exogenous gene (gene or cDNA) according to the present disclosure,
and re-infused back into the subject organism (e.g., a patient). As
a specific example, autologous cancer cells can be modified
according to some embodiments of the present invention to express
one or more immunostimulatory proteins and then may be
re-introduced into the patient, e.g., as a cancer vaccine.
[0119] One of skill in the art will recognize several advantages of
embodiments of the instant invention arising from the unexpected
high transduction and/or transgene expression efficiencies taught
herein. In particular, the use of adenoviral vectors can produce
certain advantages over lentiviral vectors. For example, adenovirus
can be engineered to carry large payloads, such that the vector
carries larger inserts of exogenous genetic material. For example,
Ad5 vectors typically have a deletion in the early transcribed
genes (E1 and/or E3) of the viral genome, where novel genetic
information can be introduced (the E1 deletion also renders the
recombinant virus replication defective). Furthermore, large
quantities of vector can be generated to transduce large quantities
of cells in a single round. As is known in the art, adenovirus
vectors are relatively easy to concentrate and purify. Moreover,
clinical studies have provided valuable information on the use of
these vectors in patients.
[0120] In addition, the improved delivery and/or expression
efficiencies require less adenovirus to achieve a given effect.
Accordingly, cytotoxicity caused by excessive exposure to
adenovirus (within the cells and/or in the culture media) can be
reduced by practicing some embodiments of the instant invention.
Additionally, where cells are transduced in a manner favoring
long-term culture, the long-term effects of transduced cells, e.g.,
expressing the desired transgene for an extended period of time,
can be analyzed. Some embodiments of the instant invention
therefore better facilitate long-term cultures and/or studies
thereof. Of particular interest is the fact that such conditions
can allow for the clinical manufacture of transgenetic material
after adenovirus transduction. For example, in some embodiments,
more that 100.sup.9, more that 100.sup.11 or more that 100.sup.13
cells can be obtained by culturing after adenovirus transduction.
Accordingly, the present invention can also be used to produce more
efficiently transgenic cells for use in cell therapy.
[0121] One of skill in the art will recognize other applications in
which enhanced introduction and/or expression of genetic
information in T cells would be desirable, in light of the detailed
disclosures provided herein.
[0122] All citations are expressly incorporated herein in their
entirety by reference.
EXPERIMENTAL
Example 1
Transduction of Anti-CD3/Anti-CD28-Activated T-Lymphocytes
A. CD3Ab/CD28Ab-activated T-cells
[0123] Primary T lymphocytes were obtained from healthy donors. In
the case of frozen samples, the cells were thawed and cultured in
RPMI medium supplemented with 10% FBS1% L-glutamine and 10 ng/mL
IL-2 (Sigma 12644) at a cell density of 1 E6 cells/mL. T
lymphocytes were immediately activated via the anti-CD3/anti-CD28
pathway with Dynalbead CD3/CD28 (Invitrogen, Carlsbad, Calif.).
Preparation and usage of the Dynalbead were performed according to
the manufacturer's protocol. Briefly, resuspended beads (75 uL of
resuspended Dynalbead per I E6 cells/mL) were washed three times
with media in an eppendorf tube. Pelleting of beads in between
washes was performed with a magnet. T lymphocyte cultures were
incubated at 37.degree. C., 5% CO.sub.2 for 15 to 48 hours.
[0124] Following incubation, activated T lymphocytes were diluted
to 3E5 to 1 E6 cells/mL. Ad5/F35 vectors were diluted with culture
medium to transduce T lymphocytes at a multiplicity of infection
(MOI) in the range of 10 to 1000 infectious units per cell. Diluted
Ad5/F35 vectors were then added directly to the T lymphocytes and
returned to the incubator. Gene delivery efficiencies were assessed
by flow cytometry (when Ad5/F35 GFP vector was used) or the Cel I
assay (when Ad5/F35 ZFN vectors were used).
[0125] As shown in FIG. 1, up to 92 and 870% of activated T-cells
were transduced with Ad5 or Ad5/35 vectors at MOIs 100 and 1000
IU/cell, with a mean fluorescent intensity (MFI) of 630 and 1780
respectively.
B. Comparison of Transduction Efficiency of Bead-Activated and IL-7
Activated T Cells
[0126] Transduction efficiencies of adenoviral vectors into
IL-7-stimulated T lymphocytes was also assessed. Briefly, primary T
cell were obtained and activated as described in Example 1, except
that 10 ng/mL of IL-7 in the T cell culture was used for activation
instead of Dynalbead CD3/CD28.
[0127] As shown in FIGS. 2A and B, at MOIs of 100 and 1000 IU/cell,
T cells activated by IL-7 yielded a transduction percentage of 55%
and 70%, with MFIs of 109 and 288. By comparison, T cells activated
by Dynalbeads were transduced at 91% and 87%, with MFIs of 910 and
1620, respectively.
C. Comparison of Transduction Efficiency of Bead-Activated and
Pha/IL2-Activated T Cells
[0128] CD4+ T cells were also activated by the addition of PHA and
IL2 or by addition of anti-CD3/anti-CD28 coated beads for 4 days.
Following activation, the cells were transduced with the Ad5/35 GFP
vector at an MOI of 30 or 100. The transduction efficiency
comparing the two activation conditions at either MOI 30 or 100 was
determined by FACS analysis measuring the percentage of cells
expressing GFP within the first 2 days after transduction.
[0129] As shown in FIG. 4, bead-activated T-cells were transduced
with higher efficiency than PHA/IL2 activated cells (example, at
MOI of 30, approximately 35% of cells stimulated by PHA expressed
GFP while 65% of those cells stimulated by anti CD3/28 expressed
GFP).
[0130] These results demonstrate that Ad5/F35 transduction
pre-stimulating-T lymphocytes with beads that are coated with
monoclonal antibodies against the CD3 and CD28 cell-surface
receptors results in 60-90% transduction efficiencies, as compared
to .about.45% transduction efficiency of PHA-activated T
lymphocytes. See, e.g., Schroers et al., supra. In addition, when
directly compared with other methods of T lymphocyte stimulation
(PHA or IL-7), T cells co-stimulated by anti-CD3/anti-CD28 bead
activation achieved a significantly higher level of non-homologous
end joining (NHEJ) events when transduced with an Ad5/F35 vector
encoding for a pair of ZFNs targeting CCR5.
Example 2
Transduction of Anti-CD3/Anti-CD28 Bead-Activated T-Lymphocytes
[0131] Peripheral blood mononuclear cells (PBMC) and CD4 T-cells
were also transduced with two different preparations (lots) of
Ad5/35 GFP vector as described in Examples 1 and 2.
[0132] As shown in FIGS. 3A and 3B, at MOIs of 100 and 300, with
either preparation, Ad5/35 GFP transduced PBMC and CD4 T cells at a
high percentage (67-91%) and MFIs (598-1408). Similar transduction
efficiencies were observed with both lots of Ad5/3.5 GFP
vector.
Example 3
Transgene Expression
[0133] The levels of transgene expression in bead-activated T cells
was also assessed. CD4 T cells were either activated by the
addition of PHA and IL2 or by addition of anti-CD3/anti-CD28 coated
beads for 4 days, and the cells were transduced with the Ad5/35 GFP
vector at an MOI of 10, 30, or 100.
[0134] Transduction efficiency was determined by measuring the
percentage of cells expressing GFP (FIGS. 5A and 6A) and the level
of transgene expression was determined by measuring the mean
fluorescence intensity of the transduced population (FIGS. 5B and
6B) for all 3 MOI conditions tested at day I (FIG. 5) and day 2
(FIG. 6) post-transduction.
[0135] To demonstrate that transgene expression was transient or
that transgene expression was not affected by some delayed response
created by the PHA/11-2 activation, GFP expression was monitored in
the MOI 30 and 100 samples for an extended period of time out to
day 14 post transduction. FIG. 7 shows the percentage of
GFP-positive cells of bead-activated T cells transduced at MOI 30
(gray squares) or MOI 100 (gray triangles) as compared to
PH-activated T cells transduced at MOI 30 (black squares) or MOI
100 (black triangles).
[0136] To also exclude the possibility that transgene expression
was affected by a reduction in cell division and dilution of the
transduced vector sequence, the rate of T cell expansion was also
monitored by measuring the population doubling rate.
[0137] As shown in FIG. 8, bead-activated T cells grew as well as
or more robustly than PHA-activated T-cells. In addition,
bead-activated T cells were transduced with adenovirus vectors at
higher efficiencies than PHA-activated T cells. See, FIG. 9.
Example 4
Cleavage of Endogenous Targets
[0138] T-cells are activated with PHA/IL2 or anti-CD3/CD28 beads
and transduced with the Ad5/F35 virus encoding GFP, the CCR5-ZFNs,
and the GR-ZFNs as described above. Cells are harvested at day 3
and day 10 post-transduction and genomic DNA isolated. The ability
of the ZFNs to cleave their respective target sites at CCR5 or GR
is analyzed by Cell assays.
[0139] To examine how many viral copies have entered the cells, the
number of viral genomes that are present per activated transduced
cell is determined using a quantitative PCR assay that measures the
number of Ad E4 genes per endogenous RNAP genes. Quantitative PCR
is performed to determine whether the activation conditions effect
transgene expression or affect the transduction efficiency (e.g.,
allowing more adenovirus particles to enter the cells).
[0140] After transduction with the GFP vector, flow cytometry is
performed to measure the number (percentage) of cells transduced.
The mean fluorescence intensity is also be determined to evaluate
whether anti-CD3/CD28 bead results in transduction or more cells
and/or increased levels of transgene expression (e.g., if the
percentage of GFP positive cells remains relatively constant, but
the mean fluorescence is increased). The difference in the level of
Ad5/F35 receptor (CD46) on the cell surface in response to
different activation conditions is also determined by flow
cytometry. To determine whether transduction is in response to
upregulation of CD46 receptors on the cell surface by anti-CD3/CD28
activation, the cells are stained using an anti-CD46 antibody and
changes in the MFI are evaluated using flow cytometry.
Example 5
Transduction with Ad5/11
[0141] To demonstrate the versatility of the method, T-cells are
prepared and activated as described in Example 1. The cells are
then transduced with an Ad5/11 vector instead of Ad5/F35. Similar
analysis is performed to verify T-cell transduction efficiency and
transgene activity.
[0142] It will be apparent to those skilled in the art that various
modifications and variations can be made to the present invention
without departing from the spirit and scope of the invention. Thus
it is intended that the present invention cover the modifications
and variations of this invention provided they come within the
scope of the appended claims and their equivalents.
[0143] All publications, articles, patents, and patent applications
mentioned in this specification are herein incorporated by
reference to the same extent as if each individual publication,
article, patent or patent application was specifically and
individually indicated as being incorporated by reference.
Sequence CWU 1
1
29112DNAArtificial SequenceDerived from human CCR-5 gene
1gatgaggatg ac 1227PRTArtificial SequenceSynthetic zinc finger
DNA-binding domain targeted to human CCR-5 gene 2Asp Arg Ser Asn
Leu Ser Arg1 537PRTArtificial SequenceSynthetic zinc finger
DNA-binding domain targeted to human CCR-5 gene 3Thr Ser Ala Asn
Leu Ser Arg1 547PRTArtificial SequenceSynthetic zinc finger
DNA-binding domain targeted to human CCR-5 gene 4Arg Ser Asp Asn
Leu Ala Arg1 557PRTArtificial SequenceSynthetic zinc finger
DNA-binding domain targeted to human CCR-5 gene 5Ile Ser Ser Asn
Leu Asn Ser1 567PRTArtificial SequenceSynthetic zinc finger
DNA-binding domain targeted to human CCR-5 gene 6Val Ser Ser Asn
Leu Thr Ser1 577PRTArtificial SequenceSynthetic zinc finger
DNA-binding domain targeted to human CCR-5 gene 7Asn Arg Asp Asn
Leu Ser Arg1 587PRTArtificial SequenceSynthetic zinc finger
DNA-binding domain targeted to human CCR-5 gene 8Thr Ser Gly Asn
Leu Thr Arg1 5912DNAArtificial SequenceDerived from human CCR-5
gene 9aaactgcaaa ag 12107PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 10Arg Ser
Asp Asn Leu Ser Val1 5117PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 11Gln Asn
Ala Asn Arg Ile Thr1 5127PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 12Arg Ser
Asp Val Leu Ser Glu1 5137PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 13Gln Arg
Asn His Arg Thr Thr1 5147PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 14Arg Ser
Asp Asn Leu Ser Asn1 5157PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 15Gln Arg
Val Asn Leu Ile Val1 5167PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 16Arg Ser
Asp Asn Leu Gly Val1 5177PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 17Gln Lys
Ile Asn Leu Gln Val1 5187PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 18Arg Ser
Asp His Leu Ser Glu1 51912DNAArtificial SequenceDerived from human
CCR-5 gene 19gacaagcagc gg 12207PRTArtificial SequenceSynthetic
zinc finger DNA-binding domain targeted to human CCR-5 gene 20Arg
Ser Ala His Leu Ser Glu1 5217PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 21Arg Ser
Ala Asn Leu Ser Glu1 5227PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 22Arg Ser
Ala Asn Leu Ser Val1 5237PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 23Asp Arg
Ala Asn Leu Ser Arg1 52413DNAArtificial SequenceDerived from human
CCR-5 gene 24catctgctac tcg 13257PRTArtificial SequenceSynthetic
zinc finger DNA-binding domain targeted to human CCR-5 gene 25Arg
Ser Asp Ser Leu Ser Lys1 5267PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 26Asp Asn
Ser Asn Arg Ile Lys1 5277PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 27Arg Ser
Ala Val Leu Ser Glu1 5287PRTArtificial SequenceSynthetic zinc
finger DNA-binding domain targeted to human CCR-5 gene 28Thr Asn
Ser Asn Arg Ile Thr1 52925PRTArtificial SequenceSynthetic C2H2
Class Zinc Finger 29Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa His Xaa Xaa Xaa Xaa Xaa His20
25
* * * * *