U.S. patent application number 09/454820 was filed with the patent office on 2001-12-13 for high efficiency genetic modification methods.
Invention is credited to KILLION, CATHERINE, KRUGER, MARK, LUNDAK, CHERYL, MCLAUGHLIN-TAYLOR, ELIZABETH.
Application Number | 20010051374 09/454820 |
Document ID | / |
Family ID | 21997910 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051374 |
Kind Code |
A1 |
MCLAUGHLIN-TAYLOR, ELIZABETH ;
et al. |
December 13, 2001 |
HIGH EFFICIENCY GENETIC MODIFICATION METHODS
Abstract
A method is provided for producing a population of genetically
modified T cells. In the method, an in vitro population of T cells
is activated by contacting said population with a CD3 binding
agent. Genetic modification is then carried out with the activated
T cells by contacting the same with a suitable gene transfer
vector.
Inventors: |
MCLAUGHLIN-TAYLOR, ELIZABETH;
(SAN CLEMENTE, CA) ; KRUGER, MARK; (ENCINITAS,
CA) ; LUNDAK, CHERYL; (SAN DIEGO, CA) ;
KILLION, CATHERINE; (LONG BEACH, CA) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
21997910 |
Appl. No.: |
09/454820 |
Filed: |
December 3, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09454820 |
Dec 3, 1999 |
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09132541 |
Aug 11, 1998 |
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6114113 |
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60055453 |
Aug 11, 1997 |
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Current U.S.
Class: |
435/455 ;
435/320.1; 435/372.3; 435/456 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12N 2740/13043 20130101; C12N 2501/515 20130101; C12N 5/0636
20130101; C12N 2800/108 20130101; C12N 15/86 20130101; C12N 15/87
20130101; C12N 2750/14143 20130101 |
Class at
Publication: |
435/455 ;
435/456; 435/320.1; 435/372.3 |
International
Class: |
C12Q 001/68; C12N
015/00; C12N 015/09; C12N 015/63; C12N 015/70; C12N 015/74; C12N
005/08; C12N 015/85; C12N 015/87; C12N 015/86 |
Claims
We claim:
1. A method for producing a population of transduced T cells, said
method comprising: (a) providing an in vitro population of T cells;
(b) activating the T cells by contacting said population with a CD3
binding agent; and (c) transducing activated T cells obtained in
step (b) by contacting said T cells with a suitable gene transfer
vector, wherein transduction is carried out when the cell density
of the T cell population is between about 0.1.times.10.sup.6 and
5.times.10.sup.6.
2. The method of claim 1, wherein transduction in step (c) is
carried out when the cell density of the T cell population is
between about 0.5.times.10.sup.6 and 2.times.10.sup.6.
3. The method of claim 1, wherein the gene transfer vector
comprises a promoter operably linked to a first nucleotide sequence
capable of being expressed to provide a transduced cell with
enhanced susceptibility to a selected cytotoxic agent.
4. The method of claim 3, wherein the first nucleotide sequence is
a suicide gene.
5. The method of claim 4, wherein the first nucleotide sequence is
a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.
6. The method of claim 1, wherein the gene transfer vector further
comprises a second nucleotide sequence encoding a selectable
marker.
7. The method of claim 6, wherein the selectable marker is capable
of providing a transduced T cell with resistance to a selected
cytotoxic agent.
8. The method of claim 7, wherein the selectable marker is neomycin
phosphotransferase II.
9. The method of claim 6, wherein the selectable marker is a cell
surface marker.
10. The method of claim 5, wherein the gene transfer vector further
comprises a selectable marker.
11. The method of claim 10, wherein the selectable marker is
neomycin phosphotransferase II.
12. The method of claim 7, further comprising a selection step
which comprises: contacting the T cells obtained after step (c)
with the selected cytotoxic agent, whereby non-transduced T cells
can be negatively selected away from the population.
13. The method of claim 9, further comprising a selection step
which comprises: contacting the T cells obtained after step (c)
with a binding molecule specific for the cell surface marker,
whereby transduced T cells can be positively selected away from the
population.
14. The method of claim 1, further comprising a selection step
which comprises fluorescence-activated cell sorting (FACS) of the T
cells obtained after step (c), whereby non-transduced T cells can
be separated from transduced T cells.
15. The method of claim 13, wherein the selection step comprises
fluorescence-activated cell sorting (FACS) of the T cells obtained
after step (c).
16. The method of claim 1, wherein the gene transfer vector is a
retroviral vector.
17. The method of claim 5, wherein the gene transfer vector is a
retroviral vector.
18. The method of claim 1, wherein the T cell population is
contacted with the CD3 binding agent in step (b) for 3 to 4
days.
19. The method of claim 1, wherein the CD3 binding agent is an
antibody molecule specific for CD3.
20. The method of claim 20, wherein the antibody molecule is an
OKT-3 antibody.
21. The method of claim 1, wherein transduction in step (c) is
carried out with a viral vector at a multiplicity of infection
(MOI) of about 3 or greater.
22. A method for producing a population of transduced T cells, said
method comprising: (a ) providing an in vitro population of T
cells; (b) activating the T cells by contacting said population
with a CD3 binding agent and a mitogen; and (c) transducing
activated T cells obtained in step (b) by contacting said T cells
with a suitable gene transfer vector, where in transduction is
carried out when the cell density of the T cell population is
between about 0.1.times.10.sup.6 and 5.times.10.sup.6.
23. The method of claim 22, wherein the mitogen in step (b) is a
cytokine.
24. The method of claim 23, wherein the cytokine is IL-2.
25. The method of claim 24, wherein the IL-2 contacted with the
population of T cells in step (b) is added to the population at a
concentration of about 50 to 100 .mu.g/mL.
26. The method of claim 22, wherein transduction in step (c) is
carried out when the cell density of the T cell population is
between about 0.5.times.10.sup.6 and 2.times.10.sup.6.
27. The method of claim 22, wherein the gene transfer vector
comprises a first nucleotide sequence capable of being expressed to
provide a transduced cell with enhanced susceptibility to a
selected cytotoxic agent.
28. The method of claim 27 wherein the first nucleotide sequence is
a suicide gene.
29. The method of claim 28, wherein the first nucleotide sequence
is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.
30. The method of claim 22, wherein the gene transfer vector is a
retroviral vector.
31. The method of claim 30, wherein the retroviral vector is added
to the T cell population in step (c) at a multiplicity of infection
(MOI) of about 3 or greater.
32. A method for producing a population of transduced T cells, said
method comprising: (a) providing an in vitro population of T cells;
(b) activating the T cells by contacting said population with a CD3
binding agent; (c) washing the T cell population obtained in step
(b) and re-seeding the T cells at a cell density of about
5.times.10.sup.5; and (d) transducing the T cell population
obtained in step (c) by contacting said T cells with a suitable
gene transfer vector, wherein transduction is carried out when the
cell density of the T cell population is between about
5.times.10.sup.5 and 2.times.10.sup.6.
33. The method of claim 32, wherein the gene transfer vector
comprises a first nucleotide sequence capable of being expressed to
provide a transduced cell with enhanced susceptibility to a
selected cytotoxic agent.
34. The method of claim 33 wherein the first nucleotide sequence is
a suicide gene.
35. The method of claim 34, wherein the first nucleotide sequence
is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.
36. The method of claim 32, wherein the gene transfer vector is a
retroviral vector.
37. A method for obtaining a transduction efficiency of 100% or
greater in a nonselected population of transduced T cells, said
method comprising: (a) providing an in vitro population of T cells;
(b) activating the T cells by contacting said population with a CD3
binding agent; (c) transducing activated T cells obtained in step
(b) by contacting said T cells with a retroviral vector at a
multiplicity of infection (MOI) of about 3 or greater, wherein
transduction is carried out when the cell density of the T cell
population is between about 5.times.10.sup.5 and
2.times.10.sup.6.
38. The method of claim 37, wherein the retroviral vector comprises
a first nucleotide sequence capable of being expressed to provide a
transduced cell with enhanced susceptibility to a selected
cytotoxic agent.
39. The method of claim 38 wherein the first nucleotide sequence is
a suicide gene.
40. The method of claim 39, wherein the first nucleotide sequence
is a Herpes Simplex Virus thymidine kinase (HSV-tk) gene.
41. A kit for producing a population of transduced T cells, said
kit comprising a CD3 binding agent contained in one or more
containers, a gene transfer vector contained in one or more
containers, ancillary reagents and/or hardware, and instructions
for use of the kit.
42. The kit of claim 41, wherein the CD3 binding agent is an
antibody molecule specific for CD3.
43. The kit of claim 42, wherein the antibody molecule is an OKT-3
antibody.
44. The kit of claim 41, wherein the gene transfer vector comprises
a promoter operably linked to a first nucleotide sequence capable
of being expressed to provide a transduced cell with enhanced
susceptibility to a selected cytotoxic agent.
45. The kit of claim 44 wherein the first nucleotide sequence is a
suicide gene.
46. The kit of claim 45, wherein the first nucleotide sequence is a
Herpes Simplex Virus thymidine kinase (HSV-tk) gene.
47. The kit of claim 41, wherein the gene transfer vector further
comprises a second nucleotide sequence encoding a selectable
marker.
48. The kit of claim 47, wherein the selectable marker is capable
of providing a transduced T cell with resistance to a selected
cytotoxic agent.
49. The kit of claim 48, wherein the selectable marker is neomycin
phosphotransferase II.
50. The kit of claim 48, wherein the selectable marker is a cell
surface marker.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to Provisional Patent
Application Serial No. 60/055,453, filed Aug. 11, 1997, from which
priority is claimed under 35 USC .sctn.119(e)(1) and which
application is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to methods for
genetically modifying a population of cells with high efficiency
and to methods of gene delivery. More particularly, the invention
relates to a method for genetically modifying a population of T
cells ex vivo.
BACKGROUND OF THE INVENTION
[0003] Gene therapy provides a method for transferring a desired
gene to a subject with the subsequent in vivo expression thereof.
Gene transfer is generally accomplished by genetically modifying
the subject's cells or tissues ex vivo, using an appropriate
vector, and reintroducing the modified cells into the host.
Alternatively, genetic material can be transferred directly into
the cells and tissues of the subject.
[0004] A number of viral based systems have been used for gene
delivery. For example, retroviral systems are known and generally
employ packaging lines which have an integrated defective provirus
(the "helper") that expresses all of the genes of the virus but
cannot package its own genome due to a deletion of the packaging
signal, known as the psi (.PSI.) sequence. Thus, the cell line
produces empty viral shells. Producer lines can be derived from the
packaging lines which, in addition to the helper, contain a viral
vector which includes sequences required in cis for replication and
packaging of the virus, known as the long terminal repeats (LTRs).
The gene of interest can be inserted in the vector and packaged in
the viral shells synthesized by the retroviral helper. The
recombinant virus can then be isolated and delivered to a subject.
(See, e.g., U.S. Pat. No. 5,219,740.)
[0005] A critical factor in achieving effective gene transfer is
the ability to obtain viral infection of a sufficient proportion of
the contacted cells. Often in gene transfers, less than one-third
of the cells contacted by a virus ex vivo are effectively modified.
Furthermore, large numbers of genetically modified cells are
required for most gene delivery applications. Thus, where the
efficiency of viral infection is low, the difficulty in obtaining a
sufficient number of genetically modified cells can present a
limiting step in achieving effective therapy. There thus exists a
need for efficient and effective genetic modification of mammalian
cells. The present invention satisfies this need and provides
related advantages as well.
SUMMARY OF THE INVENTION
[0006] A method is provided for producing a population of
genetically modified T cells. In the method, an in vitro population
of T cells is activated by contacting said population with a CD3
binding agent. Genetic modification is then carried out with the
activated T cells by contacting the same with a suitable gene
transfer vector. In the practice of the invention, genetic
modification is carried out when the cell density of the T cell
population is between about 0.1.times.10.sup.6 and
5.times.10.sup.6.
[0007] In various aspects of the invention, the gene transfer
vector comprises a promoter operably linked to a first nucleotide
sequence capable of being expressed to provide a genetically
modified cell with enhanced susceptibility to a selected cytotoxic
agent. Thus, the first nucleotide sequence can be a drug
susceptibility gene such as a Herpes Simplex Virus thymidine kinase
(HSV-tk) gene. Furthermore, the gene transfer vector can comprise a
retroviral vector containing one or more nucleotide sequences of
interest.
[0008] In another embodiment, a method is provided for obtaining a
transduction efficiency of 100% or greater in a non-selected
population of transduced T cells. The method includes the following
steps: (a) providing an in vitro population of T cells; (b)
activating the T cells by contacting the T cell population with a
CD3 binding agent; and (c) transducing the activated T cells with a
retroviral vector at a multiplicity of infection (MOI) of about 3
or greater, wherein transduction is carried out when the cell
density of the T cell population is between about 5.times.10.sup.5
and 2.times.10.sup.6.
[0009] In yet another embodiment, a kit is provided for producing a
population of transduced T cells. The kit comprises a CD3 binding
agent contained in one or more containers, a gene transfer vector
contained in one or more containers, ancillary reagents and/or
hardware, and instructions for use of the kit.
[0010] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is a map of the plasmid pLXSN-N29g.
[0012] FIG. 2 is a map of the plasmid pLXSN-T84.66g.
[0013] FIG. 3 is a map of the retroviral TK vector DAHSVTK9A.
[0014] FIG. 4 is a map of the RVV HSV-TK Provector.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of virology,
microbiology, molecular biology, recombinant DNA techniques and
immunology within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNA
Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);
Oligonucleotide Synthesis (N. Gait, ed., 1984); A Practical Guide
to Molecular Cloning (1984); Fundamental Virology, 2nd Edition,
vol. I & II (B. N. Fields and D. M. Knipe, eds.); Methods In
Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.);
and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and
C. C. Blackwell, eds., 1986, Blackwell Scientific Publications)
[0016] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
[0017] I. Definitions
[0018] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0019] "Gene transfer" or "gene delivery" refers to methods or
systems for reliably inserting DNA of interest into a host cell.
Such methods can result in transient expression of non-integrated
transferred DNA, extrachromosomal replication and expression of
transferred replicons (e.g., episomes), or integration of
transferred genetic material into the genomic DNA of host cells. "T
lymphocytes" or "T cells" are non-antibody producing lymphocytes
that constitute a part of the cell-mediated arm of the immune
system. T cells arise from immature lymphocytes that migrate from
the bone marrow to the thymus, where they undergo a maturation
process under the direction of thymic hormones. Here, the mature
lymphocytes rapidly divide increasing to very large numbers. The
maturing T cells become immunocompetent based on their ability to
recognize and bind a specific antigen. Activation of
immunocompetent T cells is triggered when an antigen binds to the
lymphocyte's surface receptors.
[0020] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell. A cell has been "transfected" when exogenous
DNA has been introduced inside the cell membrane. A number of
transfection techniques are generally known in the art. See, e.g.,
Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989)
Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
Such techniques can be used to introduce one or more exogenous DNA
moieties into suitable host cells. The term refers to both stable
and transient uptake of the genetic material, and includes uptake
of peptide- or antibody-linked DNAs.
[0021] The term "transduction" denotes the delivery of a DNA
molecule to a recipient cell either in vivo or in vitro, via a
replication-defective viral vector, such as a retroviral gene
transfer vector.
[0022] A recipient cell which has been "genetically modified" has
been transfected or transduced, either in vivo or in vitro, with a
gene transfer vector containing a DNA molecule of interest.
[0023] By "vector," "vector construct," and "gene transfer vector,"
is meant any nucleic acid construct capable of directing the
expression of a gene of interest and which can transfer gene
sequences to target cells. Thus, the term includes cloning and
expression vehicles, as well as viral vectors.
[0024] Transfer of a "suicide gene" (e.g., a drug-susceptibility
gene) to a target cell renders the cell sensitive to compounds or
compositions that are relatively nontoxic to normal cells. Moolten,
F. L. (1994) Cancer Gene Ther. 1:279-287. Examples of suicide genes
are thymidine kinase of herpes simplex virus (HSV-tk), cytochrome
P450 (Manome et al. (1996) Gene Therapy 3:513-520), human
deoxycytidine kinase (Manome et al. (1996) Nature Medicine
2(5):567-573) and the bacterial enzyme cytosine deaminase (Dong et
al. (1996) Human Gene Therapy 7:713-720). Cells which express these
genes are rendered sensitive to the effects of the relatively
nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide
(cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine
kinase) or 5-fluorocytosine (bacterial cytosine deaminase). Culver
et al. (1992) Science 256:1550-1552, Huber et al. (1994) Proc.
Natl. Acad. Sci. USA 91:8302-8306.
[0025] A "selectable marker" refers to a nucleotide sequence
included in a gene transfer vector that has no therapeutic
activity, but rather is included to allow for simpler preparation,
manufacturing, characterization or testing of the gene transfer
vector.
[0026] A "specific binding agent" refers to a member of a specific
binding pair of molecules wherein one of the molecules specifically
binds to the second molecule through chemical and/or physical
means.
[0027] A "coding sequence" or a sequence which "encodes" a selected
molecule, is a nucleic acid molecule which is transcribed (in the
case of DNA) and translated (in the case of mRNA) into a
polypeptide in vivo when placed under the control of appropriate
regulatory sequences.
[0028] A "nucleic acid molecule," or "nucleotide sequence" can
include, but is not limited to, procaryotic sequences, eucaryotic
mRNA, cDNA from eucaryotic mRNA, genomic DNA sequences from
eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
The term also captures sequences that include any of the known base
analogs of DNA and RNA.
[0029] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, a given promoter operably linked to a
coding sequence is capable of effecting the expression of the
coding sequence when the proper enzymes are present. The promoter
need not be contiguous with the coding sequence, so long as it
functions to direct the expression thereof. Thus, for example,
intervening untranslated yet transcribed sequences can be present
between the promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence.
[0030] "Recombinant" as used herein to describe a nucleic acid
molecule means a polynucleotide of genomic, cDNA, semisynthetic, or
synthetic origin which, by virtue of its origin or manipulation:
(1) is not associated with all or a portion of the polynucleotide
with which it is associated in nature; and/or (2) is linked to a
polynucleotide other than that to which it is linked in nature. The
term "recombinant" as used with respect to a protein or polypeptide
means a polypeptide produced by expression of a recombinant
polynucleotide.
[0031] II. Modes of Carrying Out the Invention
[0032] The present invention is based on the surprising discovery
that a population of T cells can be genetically modified with high
efficiency using a vector construct in ex vivo methodologies.
[0033] T cells can be isolated from peripheral blood lymphocytes
(PBLs) by a variety of procedures known to those skilled in the
art. For example, T cell populations can be "enriched" from a
population of PBLs through the removal of accessory and B cells. In
particular, T cell enrichment can be accomplished by the
elimination of non-T cells using anti-MHC class II monoclonal
antibodies. Similarly, other antibodies can be used to deplete
specific populations of non-T cells. For example, anti-Ig antibody
molecules can be used to deplete B cells and anti-MacI antibody
molecules can be used to deplete macrophages.
[0034] T cells can be further fractionated into a number of
different subpopulations by techniques known to those skilled in
the art. Two major subpopulations can be isolated based on their
differential expression of the cell surface markers CD4 and CD8.
For example, following the enrichment of T cells as described
above, CD4.sup.+ cells can be enriched using antibodies specific
for CD4 (see Coligan et al., supra). The antibodies may be coupled
to a solid support such as magnetic beads. Conversely, CD8+ cells
can be enriched through the use of antibodies specific for CD4 (to
remove CD4.sup.+ cells), or can be isolated by the use of CD8
antibodies coupled to a solid support. CD4 lymphocytes from HIV-1
infected patients can be expanded ex vivo, before or after
transduction as described by Wilson et. al. (1995) J. Infect. Dis.
172:88.
[0035] Following purification of T cells, a variety of methods of
genetic modification known to those skilled in the art can be
performed using non-viral or viral-based gene transfer vectors
constructed as described herein. For example, one such approach
involves transduction of the purified T cell population with
vector-containing supernatant cultures derived from vector
producing cells. A second approach involves co-cultivation of an
irradiated monolayer of vector-producing cells with the purified T
cells. A third approach involves a similar co-cultivation approach;
however, the purified T cells are pre-stimulated with various
cytokines and cultured 48 hours prior to the co-cultivation with
the irradiated vector producing cells. Pre-stimulation prior to
such transduction increases effective gene transfer (Nolta et al.
(1992) Exp. Hematol. 20:1065). While not wishing to be bound by a
particular theory, the increased level of transduction is
attributed to increased proliferation of the T cells necessary for
efficient retroviral transduction. Stimulation of these cultures to
proliferate also provides increased cell populations for
re-infusion into the patient. Subsequent to co-cultivation, T cells
are collected from the vector producing cell monolayer, expanded,
and frozen in liquid nitrogen.
[0036] Gene transfer vectors, containing one or more coding
sequences of interest associated with appropriate control elements
for delivery to the isolated T cells, can be assembled using known
methods.
[0037] Selectable markers can also be used in the construction of
gene transfer vectors. For example, a marker can be used which
imparts to a mammalian cell transduced with the gene transfer
vector resistance to a cytotoxic agent. The cytotoxic agent can be,
but is not limited to, neomycin, aminoglycoside, tetracycline,
chloramphenicol, sulfonamide, actinomycin, netropsin, distamycin A,
anthracycline, or pyrazinamide. For example, neomycin
phosphotransferase II imparts resistance to the neomycin analogue
geneticin (G418).
[0038] Non-immunogenic selectable markers are preferred for use
herein. "Non-immunogenic" refers to a selectable marker or prodrug
activating enzyme that does not cause an undesired immune reaction
in the majority of patients when it is administered as part of a
gene delivery vehicle. Such genes may be human genes, non-human
genes, or mutated human genes that lack sufficient difference from
normal human genes (normally less than 10% amino acid sequence
difference). Genes that are not of human origin for use herein will
not carry epitopes that allow effective presentation of the protein
sequence through MHC class I or class II presentation in patients,
or may be genes that carry sequences that prevent the effective
presentation of otherwise immunogenic epitopes. It is important to
note that at least some non-immunogenic selectable markers will be
species-specific. For clinical use, non-immunogenic markers will
generally be of human origin.
[0039] A wide variety of non-immunogenic markers may be expressed
by the gene transfer vectors of the present invention. Briefly,
such markers may be readily tested for immunogenicity by a variety
of assays, including for example, CTL assays for antigens to which
the organism has previously generated immunity, and in vitro
generation of T-cell response utilizing dendritic cells transduced
with the antigen or antigens to which the organism does not have a
previously existing response (see Henderson et al. (1996) Canc.
Res. 56:3763; Hsu et al. (1995) Nat. Med. 2:52). CTL assays can be
conducted as described in, e.g., International Publication Number
WO 91/02805. Another method for ensuring that a marker is
non-immunogenic is to administer the marker in a standard skin test
such as one utilized to test allergic reactions. It should be noted
however, that while the above tests may be utilized in order to
ascertain markers which are non-immunogenic within the context of
the present invention, a small percentage of patients may
nevertheless react against the markers.
[0040] Suitable non-immunogenic markers may be obtained from a
variety of sources. For example, the marker may be, in its native
state, a human enzyme, and thus, by its very nature,
non-immunogenic. Similarly, markers from closely related species
such as macaques may likewise be non-immunogenic. The marker may be
of non-human origin, and can be made non-immunogenic by mutation
(e.g., substitution, deletion or insertion). Representative
examples of such markers and associated prodrug molecules include
alkaline phosphatase and various toxic phosphorylated compounds
such as phenolmustard phosphate, doxorubicin phosphate, mitomycin
phosphate and etoposide phosphate; .beta.-galactosidase and
N-[4-(.beta.-D-galactopyranosyl) benyloxycarbonyl]-daunorubicin;
azoreductase and azobenzene mustards; .beta.-glucosidase and
amygdalin; .beta.-glucuronidase and phenolmustard-glucuronide and
epirubicin-glucuronide; carboxypeptidase A and
methotrexate-alanine; cytochrome P450 and cyclophosphamide or
ifosfamide; DT diaphorase and
5-(aziridine-1-yl)-2,4,dinitrobenzamide (CB1954) (Cobb et al.
(1969) Biochem. Pharmacol 18:1519, Knox et al. (1993) Cancer
Metastasis Rev. 12:195); .beta.-glutamyl transferase and
.beta.-glutamyl p-phenylenediamine mustard; nitroreductase and
CB1954 or derivatives of 4-nitrobenzyloxycarbonyl; glucose oxidase
and glucose; xanthine oxidase and hypoxanthine; and plasmin and
peptidyl-p-phenylenediamine-mustard. Non-immunogenic markers may
also be made by expressing an enzyme in a compartment of the cell
where it is not normally expressed. For example, the enzyme furin,
normally expressed in the trans-Golgi, can be expressed on the cell
surface. It can then activate drugs that normally may not reach the
trans-Golgi.
[0041] Alternatively, the exogenous selectable marker can be a
protein which is expressed on the surface of a cell such that cells
expressing the marker can be physically separated from other cells
in a population by immunochemical or receptor-ligand binding
methods. Cell surface markers can include cell-adhesive factors,
such as the integrins, which modulate cell binding to extracellular
matrix proteins.
[0042] In one particular embodiment, vectors expressing a suicide
gene are provided. Coding sequences for a suicide gene can be
obtained using known methods. For example, the coding region and
transcriptional termination signals of HSV-I thymidine kinase gene
(HSV-TK) can be isolated from plasmid 322TK (McKnight et. al.
(1980) Nuc. Acids Res. 8:5949) and then cloned into a suitable gene
transfer vector.
[0043] In other embodiments, vectors expressing human Factor VIII
and IX can be provided for use in the treatment of hemophilia.
Particularly, vectors expressing a B domain-deleted factor VIII
protein are described in the examples below. The B domain separates
the second and third A domains of factor VIII in the newly
synthesized single-chain molecule. The B domain extends from amino
acids 712 to 1648 of the molecule. Wood et al. (1984) Nature
312:330-337. Proteolytic activation of factor VIII involves
cleavage at specific Arg residues located at positions 372, 740,
and 1689. Cleavage of plasma factor VIII by thrombin or Factor Xa
at Arg 372 and Arg 1689 are essential for obtaining active factor
VIII. Activated factor VIII consists of a calcium-bridged
heterodimer comprising amino acids residues 1-372 (containing the
A1 domain) and residues 373-740 (containing the A2 domain), and
residues 1690-2332 (containing the A3-C1-C2 domain).
[0044] An important advantage in using a B domain-deleted factor
VIII molecule in the practice of the invention is that the reduced
size appears to be less prone to proteolytic degradation and,
therefore, no addition of plasma-derived albumin is necessary for
stabilization of the final product. The term "B domain deletion" as
used herein with respect to factor VIII protein refers to a factor
VIII protein in which some or all of the amino acids between
residues 711 and 1694 have been deleted, and which still preserves
a biologically active factor VIII molecule.
[0045] A range of B domain deletions can exist depending on which
amino acid residues in the B domain are deleted. One specific B
domain deletion, termed "the SQN deletion," exists and has been
created by fusing Ser 743 to Gln 1638 (Lind et al. (1995) Eur. J.
Biochem. 323:19-27, and International Publication No. WO 91/09122).
This removes amino acid residues 744 to 1637 from the B domain
creating a Ser-Glu-Asn (SQN) link between the A2 and A3 factor VIII
domains. When compared to plasma-derived factor VIII, the SQN
deletion does not influence the in vivo pharmacokinetics of the
factor VIII molecule (Fijnvandraat et. al. (1997) P.R.Schattauer
Vertagsgesellschatt mbH (Stuttgart) 77:298-302). The terms "Factor
VIII SQN deletion" or "SQN deletion" as used herein refer to this
deletion and to other deletions which preserve the single S-Q-N
tripeptide sequence and which result in the deletion of the amino
acids between the two B-domain SQN sequences (See International
Publication No. WO 91/09122 for a description of this amino acid
sequence).
[0046] There are number of other B domain-deleted forms of factor
VIII. cDNA's encoding all of these B domain-deleted factor VIII
proteins can be inserted into gene transfer vectors using standard
molecular biology techniques. For example, cDNA molecules encoding
the following factor VIII B domain-deletions can be employed in the
practice of the invention: des 797-1562 (Eaton (1986) Biochemistry
25:8343); des 760-1639 (LA-FVIII) (Toole (1986) Proc. Natl. Acad.
Sci. USA 83:5939); des 771-1666 (FVIII del II: missing one thrombin
site) (Meutien (1988) Prot. Eng. 2:301); des 747-1560 (Sarver
(1987) DNA 6:553); des 868-1562 and des 713-1637 (thrombin
resistant) (Mertens (1993) Br. J. Haematol. 85:133); des 797-1562
(Esmon (1990) Blood 76:1593); des 741-1668 (Donath (1995) Biochem.
J. 312:49); des 748-1648 (partially processed), des 753-1648
(partially processed), des 777-1648 (partially processed), des
744-1637 (FVIII-SQ), des 748-1645 (FVIII-RH), des B-domain +0, 1, 2
Arg (partially processed), desB +3Arg (FVIIIR4), desB +4Arg
(FVIIIR5) (Lind (1995) Eur. J. Biochem. 232:19); des 741-1689 or
des 816-1598 (Langner (1988) Behring Inst Mitt 16-25); des 746-1639
(Cheung (1996) Blood 88:325a); and des 795-1688 (thrombin sites
mutated) (Pipes (1996) Blood 88:441a).
[0047] Other factor VIII B domain deletions that can be employed
herein include, but are not limited to, a B domain deletion in
which an IgG hinge region has been inserted (see, e.g., U.S. Pat.
No. 5,595,886), and the B domain-deleted factor VIII molecules
described in commonly owned U.S. patent application entitled
"Methods for Administration of Recombinant Delivery Vehicles for
Treatment of Hemophilia and Other Disorders," filed Jun. 4, 1997 as
attorney docket No. 1155.004, which application is incorporated
herein by reference in its entirety.
[0048] The full-length factor VIII cDNA can also be inserted into
the gene transfer vectors of the invention, such as the cDNA
molecule described in International Publication No. WO 96/21035
which is hereby incorporated by reference in its entirety. A
variety of Factor VIII deletions, mutations, and polypeptide
analogs of Factor VIII also suitable for use herein include, for
example, those analogs described in International Publication Nos.
WO 97/03193, WO 97/03194, WO 97/03195, and WO 97/03191, all of
which are hereby incorporated by reference.
[0049] Hemophilia B can also be treated using gene delivery
techniques with factor IX-expressing gene transfer vectors. Human
factor IX deficiency (Christmas disease or Hemophilia B) affects
primarily males because it is transmitted as a sex-linked recessive
trait. It affects about 2000 people in the U.S. The human factor IX
gene codes for a mature protein of 416 amino acid residues.
[0050] Human factor IX cDNA can be obtained, for example, from the
plasmid construct pHfIX1 as described by Kurachi et al. (1982)
Proc. Natl. Acad. Sci. USA 79:6461-6464. The cDNA sequence can be
excised as a PstI fragment of about 1.5 kb and blunt-ended using T4
DNA polymerase. The factor cDNA fragment can then be readily
inserted, for example into a suitable restriction site in a
vector.
[0051] The present invention also can be used in therapy and/or
prophylaxis of thrombosis due to APC resistance, and other
disorders of thrombosis and hypercoagulation, by providing gene
transfer vectors capable of expressing factor V. Blood coagulation
consists of a series of sequential activations of circulating
serine protease zymogens, culminating in the activation of
prothrombin to form thrombin and the subsequent generation of
fibrin, the substance of the clot. Two of these reactions, the
activation of prothrombin and factor X, require participation of
the large proteinaceous cofactors, factors Va and VIIIa,
respectively. The serine protease zymogen (Protein C) exerts
anticoagulant effect when it is cleaved by thrombin to form
activated protein C. Activated protein C (APC) destroys the
activity of factors Va and VIIIa through cleavage at specific
arginine residues. Genetic deficiencies in protein C or its
cofactor, protein S, account for .about.5-10% of cases of familial
thrombophilia. In 1993, Dahlback described a new form of
thrombophilia, called activated protein C resistance (APC
resistance) in which added APC failed to prolong the clotting times
of patients' plasmas. Dahlback (1993) Proc. Natl. Acad. Sci. USA
90:1004. This was subsequently shown to account for up to 40% of
the cases of familial thrombophilia, making it the most common form
of inherited disposition to thrombosis (Sun et al. (1994) Blood
83:3120). Greater than 95% of APC resistance cases result from a
single point mutation in factor V, R506Q (Bertina et al. (1994)
Nature 369:64, Greengard et al. (1994) Lancet 343:1361). This
mutation was subsequently found to be present in various healthy
European populations at a level of 1-10% (Svensson et al. (1994)
New Engl. J. Med. 300:517, Griffin et al. (1993) Blood 82:1989,
Koster et al. Lancet 342:1503), and presence/absence of symptoms
can vary considerably in a family with numerous homozygotes
(Greengard et al. (1995) New Engl. J. Med. 331:1559), underscoring
the multifactorial nature of thrombotic disease. Rosendaal et al.
(1995) Blood 85:1504, estimated the relative risk of thrombosis in
a heterozygote for APC resistance as seven-fold, and for
homozygotes as 80-fold.
[0052] Greengard et al. (1995) Thromb Haemostas 73:1361(abs)
described carrying both a null allele for factor V deficiency and
APC resistance. Since these two factor V defects assorted
independently, they represent two different factor V alleles. The
compound heterozygotes had circulating factor V derived only from
the APC resistant factor V allele, and two of the three symptomatic
family members had this "pseudohomozygous" genotype. Other family
members with only factor V deficiency had no thrombosis. While not
wishing to be bound by theory, the risk factor of an APC resistance
allele can be compensated in some cases by the mere presence of
some normal (APC responsive) factor V. Thus, delivery of normal
factor V can be of therapeutic benefit even in the presence of the
same amount of resistant factor V, perhaps due to this
mechanism.
[0053] Thus, nucleotide sequences encoding factor V can be
incorporated into a gene transfer vector according to the
invention. Factor V cDNA can be obtained from pMT2-V (Jenny (1987)
Proc. Natl. Acad. Sci. USA 84:4846, ATCC Deposit No. 40515) by
digestion with SalI, and the 7 kb cDNA band excised from agarose
gels and cloned into vectors, using standard molecular biology
techniques. Either a full-length factor V cDNA, or a B domain
deletion or B domain substitution thereof, can be used. B domain
deletions of factor V, such as those reported by Marquette (1995)
Blood 86:3026 and Kane (1990) Biochemistry 29:6762, can be made as
described by the authors.
[0054] Gene transfer vectors can likewise be constructed herein to
express antithrombin III for treatment or prophylaxis of
hypercoagulable conditions. The central enzyme of the coagulation
pathways, thrombin, acts directly through cleavage of fibrinogen to
form fibrin, the substance of the clot, or indirectly through
positive feedback mechanisms involving activation of other clotting
factors. The most commonly used acute-phase anticoagulant used is
heparin which augments thrombin inhibition. The major thrombin
inhibitor in plasma is antithrombin III (ATIII). The frequency of
ATIII deficiency is as high as 1:500 (Tait (1990) Br. J. Haematol.
75:141). Although most cases are clinically silent, deficiency may
pose a risk factor synergistic with others. Most patients are
treated with oral anticoagulants supplemented by ATIII concentrates
for surgery or other major trauma (Winter (1981) Br. J. Haematol.
49:449-457). Oral anticoagulation is considered an inconvenient and
inadequate treatment for hypercoagulable states, while
plasma-derived proteins carry the risk of transmittal of infectious
agents and other problems. Acquired deficiencies of ATIII are more
frequent, such as in premature infants, L-asparaginase therapy for
leukemia, DIC, sepsis, nephrotic syndromes, traumatic bleeding,
severe burns, malignancies, ARDS, DVT/PE, and enteropathies.
Concentrates have been used for animal models of some of these
conditions (Emerson (1994) Blood Coag. Fibrinol. 5:37). The use of
gene therapy to deliver ATIII using the methods described herein
can provide useful therapy, particularly in ATIII deficiency
states.
[0055] Gene transfer vectors expressing ATIII can thus be
constructed from the vector pKT218 (Prochownik (1983) J. Biol.
Chem. 258:8389, ATCC Deposit No. 57224/57225) by excision with
PstI. The 1.6 kb cDNA insert can be recovered from agarose gels and
cloned into a suitable viral or non-viral vector system.
[0056] As described above, protein C is a serine protease zymogen
that acts to downregulate the coagulation cascade. Protein C
deficiency is associated with increased risk of recurrent
thrombosis, purpura fulminans, and warfarin-induced skin necrosis
(Bauer, Disorders of Hemostasis, Ratnoff & Forbes (Eds), W B
Saunders, Philadelphia (1996)). The incidence of heterozygosity is
as high as 1/200 (Miletich (1987) New Engl. J. Med. 317:991).
Although most cases are clinically silent, deficiency may pose a
risk factor synergistic with others. Recombinant protein C is
administered on a compassionate basis to severely affected
homozygotes (Minford (1996) Br. J. Haematol. 93:215). Homozygotes
and symptomatic heterozygotes could be treated more effectively by
gene delivery techniques. In addition, there is evidence to suggest
that augmenting levels of activated protein C (APC) could play a
major role in prevention of thrombosis in patients with other
causes (genetic or acquired) of hypercoagulability. In this regard,
Gruber (1992) Blood 79:2340, showed that low levels of APC
circulate in the plasma of normals, and proposed that basal levels
of APC serve to downregulate coagulation in response to low-level
prothrombotic signals. The ratio of circulating endogenous APC
level to protein C zymogen level was lower in protein C-deficient
individuals with a history of thrombosis, than in thrombosis-free
relatives, but APC levels are generally proportional to zymogen
protein C levels (Espana (1996) Thrombos Haemostas 75:56-61). While
not wishing to be bound by a particular theory, it may be that gene
therapy vectors which express protein C in non-deficient
individuals at risk for thrombosis from other causes will have a
protective effect in individuals with normal levels of protein C
due to this mechanism. An artificial variant of protein C,
HPC-FLINQ (Richardson (1992) Nature 360:261, Kurz (1997) Blood
89:534) was recently described with an enhanced activation profile
in the presence of thrombin without the normally required cofactor,
thrombomodulin, so that APC was generated in the presence of
thrombin levels attained during the clotting of plasma. In
addition, HPC-S460A, a second artificial variant of human protein
C, has a normal activation profile but a much lowered propensity
for subsequent inhibition by plasma serpins. While not wishing to
be bound by theory, since binding to serpins is the major mechanism
for removal of APC from the circulation, the nonenzymatic
anticoagulant activity demonstrated for this variant (Gale (1997)
Prot. Sci. 6:132) may be preferred due to its having a
significantly prolonged plasma half-life upon activation. Yet
another approach was taken by Ehrlich (1989) J. Biol. Chem.
264:14298, who made a variant of protein C that would became
activated during the process of secretion, resulting in secretion
of the activated enzyme. In particular, delivery of these variants
by the means of gene transfer vectors and the genetic modification
methods described herein are useful in reducing thrombosis in
individuals at risk thereof.
[0057] Thus, gene transfer vectors capable of expressing Protein C
can be made using techniques known to those of skill in the art.
For example, protein C cDNA can be obtained by restriction enzyme
digestion of known vectors containing the same (Foster (1984) Proc.
Natl. Acad. Sci. USA 81:4766, Beckmann (1985) Nucleic Acids Res.
13:5233). The 1.6 kb cDNA insert can then be recovered from agarose
gels and cloned into suitable cloning sites of viral and non-viral
vectors under standard conditions.
[0058] The normal protein C anticoagulant pathway requires
activation by the enzyme thrombin. Thrombin is normally a
procoagulant enzyme which cleaves fibrinogen to form fibrin,
activates platelets, and performs positive feedback reactions on
components of the coagulation cascade. Thrombin activity in the
anticoagulant pathway under physiological conditions is dependent
upon binding to an endothelial cell surface-bound cofactor,
thrombomodulin. Upon binding to this protein, thrombin undergoes a
conformational change that greatly reduces it's ability to perform
the procoagulant reactions mentioned above, while greatly
increasing the rate of activation of protein C zymogen, thus
changing specificity from a procoagulant to an anticoagulant
enzyme. In accordance with this model, infusion of low levels of
thrombin has been shown to be antithrombotic (Gruber (1990) Circ.
82:578, Hanson (1993) J. Clin. Invest. 92:2003, McBane (1995)
Thromb. Haemostas. 74:879). Thrombin variants with similar changes
in specificity in the absence of thrombomodulin have been developed
(Dang (1997) Nature Biotech. 15:146, Gibbs (1995) Nature 378:413,
(1991) Proc. Natl. Acad. Sci. USA 88:7371, Wu (1991) Proc. Natl.
Acad. Sci. USA 88:6775, and Guinto (1995) Proc. Natl. Acad. Sci.
USA 92:11185). Delivery of these variants by the means of gene
transfer vectors and the methods of genetic modification described
herein is thus useful in reducing thrombosis in individuals at risk
thereof.
[0059] Gene transfer vectors expressing prothrombin and its
variants can be constructed by methods known to those of skill in
the art. For example, prothrombin cDNA can be obtained by
restriction enzyme digestion of a published vector (Degen (1983)
Biochemistry 22:2087). The 1.9 kb cDNA insert can then be recovered
from agarose gels and cloned into a suitable vector using the
techniques described herein.
[0060] Finally, the endothelial cell surface protein,
thrombomodulin, is a necessary cofactor for the normal activation
of protein C by thrombin. A soluble recombinant form has been
described (Parkinson (1990) J. Biol. Chem. 265:12602), which was
proposed for use as a clinical therapeutic anticoagulant acting via
the protein C pathway. Delivery of this and other variants by the
gene transfer vectors and genetic modification methodology of the
present invention is therefore useful in reducing thrombosis in
individuals at risk.
[0061] Gene transfer vector expressing thrombomodulin and its
variants can be constructed using techniques known to those of
skill in the art. In this regard, thrombomodulin cDNA can be
obtained from the vector puc19TM15 (Jackman (1987) Proc. Natl.
Acad. Sci. USA 84:6425, Shirai (1988) J. Biochem. 103:281, Wen
(1987) Biochemistry 26:4350, Suzuki (1987) EMBO J. 6:1891, ATCC
Deposit No. 61348, 61349) by excision with SalI. The 3.7 kb cDNA
insert can be recovered from agarose gels and cloned into a
suitable viral or non-viral vector system.
[0062] There are a number of proteins useful for treatment of
hereditary disorders that can be expressed in vivo by the methods
of invention. Many genetic diseases caused by inheritance of
defective genes result in the failure to produce normal gene
products, for example, thalassemia, phenylketonuria, Lesch-Nyhan
syndrome, severe combined immunodeficiency (SCID), hemophilia A and
B, cystic fibrosis, Duchenne's Muscular Dystrophy, inherited
emphysema and familial hypercholesterolemia (Mulligan et al. (1993)
Science 260:926, Anderson et al. (1992) Science 256:808, Friedman
et al. (1989) Science 244:1275). Although genetic diseases may
result in the absence of a gene product, endocrine disorders, such
as diabetes and hypopituitarism, are caused by the inability of the
gene to produce adequate levels of the appropriate hormone insulin
and human growth hormone respectively.
[0063] Gene therapy by the methods of the invention is a powerful
approach for treating these types of disorders. This therapy
involves the introduction of normal recombinant genes into T cells
so that new or missing proteins are produced by the T cells after
introduction or reintroduction thereof into a patient. A number of
genetic diseases have been selected for treatment with gene
therapy, including adenine deaminase deficiency, cystic fibrosis,
.alpha..sub.1-antitrypsin deficiency, Gaucher's syndrome, as well
as non-genetic diseases.
[0064] In particular, Gaucher's syndrome is a genetic disorder
characterized by a deficiency of the enzyme glucocerebrosidase.
This enzyme deficiency leads to the accumulation of
glucocerebroside in the lysosomes of all cells in the body. For a
review see Science 256:794 (1992) and Scriver et al., The Metabolic
Basis of Inherited Disease, 6th ed., vol. 2, page 1677). Thus, gene
transfer vectors that express glucocerebrosidase can be constructed
for use in the treatment of this disorder. Likewise, gene transfer
vectors encoding lactase can be used in the treatment of hereditary
lactose intolerance, those expressing AD can be used for treatment
of ADA deficiency, and gene transfer vectors encoding
.alpha..sub.1-antitrypsin can be used to treat
.alpha..sub.1-antitrypsin deficiency. See Ledley, F. D. (1987) J.
Pediatrics 110:157-174, Verma, I. (Nov. 1987) Scientific American
pp. 68-84, and International Publication No. WO 95/27512 entitled
"Gene Therapy Treatment for a Variety of Diseases and Disorders,"
for a description of gene therapy treatment of genetic
diseases.
[0065] Another genetic disorder, familial hypercholesterolemia, is
characterized: clinically by a lifelong elevation of low density
lipoprotein (LDL), the major cholesterol-transport lipoprotein in
human plasma; pathologically by the deposition of LDL-derived
cholesterol in tendons, skin and arteries leading to premature
coronary heart disease; and genetically by autosomal dominant
inherited trait. In heterozygotes (occurring in about 1 in 500
persons worldwide), cells are able to bind cholesterol at about
half the rate of normal cells. Heterozygote plasma cholesterol
levels show two-fold elevation starting at birth. Homozygotes occur
at a frequency of about 1/1 million persons. These individuals have
severe cholesterolemia with death occurring usually before age 20.
The disease associated with this disorder (Arteriosclerosis)
depends on geography, and affects 15.5 per 100,000 individuals in
the U.S. (20,000 total) and 3.3 per 100,000 individuals in Japan.
Gene transfer vectors expressing the LDL receptor for treatment of
disorders manifesting with elevated serum LDL can thus be
constructed by techniques known to those of skill in the art.
[0066] In still further embodiments of the invention, nucleotide
sequences which can be incorporated into a gene transfer vector
include, but are not limited to, proteins associated with
enzyme-deficiency disorders, such as the cystic fibrosis
transmembrane regulator (see, for example, U.S. Pat. No. 5,240,846
and Larrick et al. (1991) Gene Therapy Applications of Molecular
Biology, Elsevier, New York and adenosine deaminase (ADA) (see U.S.
Pat. No. 5,399,346); growth factors, or an agonist or antagonist of
a growth factor (Bandara et al. (1992) DNA and Cell Biology,
11:227); one or more tumor suppressor genes such as p53, Rb, or
C-CAMI (Kleinerman et al. (1995) Cancer Research 55:2831); a
molecule that modulates the immune system of an organism, such as a
HLA molecule (Nabel et al. (1993) Proc. Natl. Acad. Sci. USA
90:11307); a ribozyme (Larsson et al. (1996) Virology 219:161); a
peptide nucleic acid (Hirshman et al. (1996) J. Invest. Med.
44:347); an antisense molecule (Bordier et al. (1995) Proc. Natl.
Acad. Sci. USA 92:9383) which can be used to down-regulate the
expression or synthesis of aberrant or foreign proteins, such as
HIV proteins or a wide variety of oncogenes such as p53 (Hesketh,
The Oncogene Facts Book, Academic Press, New York, (1995); a
biopharmaceutical agent or antisense molecule used to treat
HIV-infection, such as an inhibitor of p24 (Nakashima et al. (1994)
Nucleic Acids Res. 22:5004); or reverse-transcriptase (see,
Bordier, supra).
[0067] Other proteins of therapeutic interest can be expressed in
vivo by gene transfer vectors using the methods of the invention.
For instance sustained in vivo expression of tissue factor
inhibitory protein (TFPI) is useful for treatment of conditions
including sepsis and DIC and in preventing reperfusion injury. (See
International Publications Nos. WO 93/24143, WO 93/25230 and WO
96/06637). Nucleic acid sequences encoding various forms of TFPI
can be obtained, for example, as described in U.S. Pat. Nos.
4,966,852; 5,106,833; and 5,466,783, and incorporated into the gene
transfer vectors described herein.
[0068] Erythropoietin (EPO) and leptin can also be expressed in
vivo from genetically modified T cells according to the methods of
the invention. For instance EPO is useful in gene therapy treatment
of a variety of disorders including anemia (see International
Publication No. WO 95/13376 entitled "Gene Therapy for Treatment of
Anemia"). Sustained delivery of leptin by the methods of the
invention is useful in treatment of obesity. See International
Publication No. WO 96/05309 for a description of the leptin gene
and the use thereof in the treatment of obesity.
[0069] A variety of other disorders can also be treated by the
methods of the invention. For example, sustained in vivo systemic
production of apolipoprotein E or apolipoprotein A from genetically
modified T cells can be used for treatment of hyperlipidemia (see
Breslow et al. (1994) Biotechnology 12:365). Sustained production
of angiotensin receptor inhibitor (Goodfriend et al. (1996) N.
Engl. J. Med. 334:1469) can be provided by the methods described
herein. As yet an additional example, the long term in vivo
systemic production of angiostatin is useful in the treatment of a
variety of tumors. (See O'Reilly et al. (1996) Nature Med.
2:689).
[0070] In other embodiments, the present gene transfer vectors can
be constructed to encode a cytokine or other immunomodulatory
molecule. For example, nucleic acid sequences encoding native IL-2
and gamma-interferon can be obtained as described in U.S. Pat. Nos.
4,738,927 and 5,326,859, respectively, while useful muteins of
these proteins can be obtained as described in U.S. Pat. No.
4,853,332. Nucleic acid sequences encoding the short and long forms
of mCSF can be obtained as described in U.S. Pat. Nos. 4,847,201
and 4,879,227, respectively. In particular aspects of the
invention, retroviral vectors expressing cytokine or
immunomodulatory genes can be produced as described herein and in
International Application No. PCT US 94/02951, entitled
"Compositions and Methods for Cancer Immunotherapy."
[0071] Examples of suitable immunomodulatory molecules for use
herein include the following: IL-1 and IL-2 (Karupiah et al. (1990)
J. Immunology 144:290-298, Weber et al. (1987) J. Exp. Med.
166:1716-1733, Gansbacher et al. (1990) J. Exp. Med. 172:1217-1224,
and U.S. Pat. No. 4,738,927); IL-3 and IL-4 (Tepper et al. (1989)
Cell 57:503-512, Golumbek et al. (1991) Science 254:713-716, and
U.S. Pat. No. 5,017,691); IL-5 and IL-6 (Brakenhof et al. (1987) J.
Immunol. 139:4116-4121, and International Publication No. WO
90/06370); IL-7 (U.S. Pat. No. 4,965,195); IL-8, IL-9, IL-10,
IL-11, IL-12, and IL-13 (Cytokine Bulletin, Summer 1994); IL-14 and
IL-15; alpha interferon (Finter et al. (1991) Drugs 42:749-765,
U.S. Pat. Nos. 4,892,743 and 4,966,843, International Publication
No. WO 85/02862, Nagata et al. (1980) Nature 284:316-320,
Familletti et al. (1981) Methods in Enz. 78:387-394, Twu et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2046-2050, and Faktor et al.
(1990) Oncogene 5:867-872); beta-interferon (Seif et al. (1991) J.
Virol. 65:664-671); gamma-interferons (Radford et al. (1991) The
American Society of Hepatology 20082015, Watanabe et al. (1989)
Proc. Natl. Acad. Sci. USA 86:9456-9460, Gansbacher et al. (1990)
Cancer Research 50:7820-7825, Maio et al. (1989) Can. Immunol.
Immunother. 30:34-42, and U.S. Pat. Nos. 4,762,791 and 4,727,138);
G-CSF (U.S. Pat. Nos. 4,999,291 and 4,810,643); GM-CSF
(International Publication No. WO 85/04188); tumor necrosis factors
(TNFs) (Jayaraman et al. (1990) J. Immunology 144:942-951); CD3
(Krissanen et al. (1987) Immunogenetics 26:258-266); ICAM-1 (Altman
et al. (1989) Nature 338:512-514, Simmons et al. (1988) Nature
331:624-627); ICAM-2, LFA-1, LFA-3 (Wallner et al. (1987) J. Exp.
Med. 166:923-932); MHC class I molecules, MHC class II molecules,
B7.1- .3, .beta..sub.2-microglobulin (Parnes et al. (1981) Proc.
Natl. Acad. Sci. USA 78:2253-2257); chaperones such as calnexin;
and MHC-linked transporter proteins or analogs thereof (Powis et
al. (1991) Nature 354:528-531). Immunomodulatory factors may also
be agonists, antagonists, or ligands for these molecules. For
example, soluble forms of receptors can often behave as antagonists
for these types of factors, as can mutated forms of the factors
themselves.
[0072] Nucleic acid molecules that encode the above-described
substances, as well as other nucleic acid molecules that are
advantageous for use within the present invention, may be readily
obtained from a variety of sources, including, for example,
depositories such as the American Type Culture Collection (ATCC,
Rockville, Md.), or from commercial sources such as British
Bio-Technology Limited (Cowley, Oxford England). Representative
examples include BBG 12 (containing the GM-CSF gene coding for the
mature protein of 127 amino acids), BBG 6 (which contains sequences
encoding gamma interferon), ATCC Deposit No. 39656 (which contains
sequences encoding TNF), ATCC Deposit No. 20663 (which contains
sequences encoding alpha-interferon), ATCC Deposit Nos. 31902,
31902 and 39517 (which contain sequences encoding beta-interferon),
ATCC Deposit No. 67024 (which contains a sequence which encodes
Interleukin-lb), ATCC Deposit Nos. 39405, 39452, 39516, 39626 and
39673 (which contain sequences encoding Interleukin-2), ATCC
Deposit Nos. 59399, 59398, and 67326 (which contain sequences
encoding Interleukin-3), ATCC Deposit No. 57592 (which contains
sequences encoding Interleukin-4), ATCC Deposit Nos. 59394 and
59395 (which contain sequences encoding Interleukin-5), and ATCC
Deposit No. 67153 (which contains sequences encoding
Interleukin-6).
[0073] Plasmids containing cytokine genes or immunomodulatory genes
can be digested with appropriate restriction enzymes, and DNA
fragments containing the particular gene of interest can be
inserted into the gene transfer vector using standard molecular
biology techniques. (See, e.g., Sambrook et al., supra., or Ausbel
et al. (eds) Current Protocols in Molecular Biology, Greene
Publishing and Wiley-Interscience, New York (1987)). In particular,
retroviral vectors expressing cytokine and immunomodulatory
molecules can be constructed as described in International
Publication Nos. WO 94/02951 and WO 96/21015, both of which are
incorporated by reference in their entirety.
[0074] A variety of known polypeptide hormones and growth factors
can also be used in the instant gene transfer vectors to provide
for therapeutic long-term expression of these proteins. Exemplary
hormones, growth factors and other proteins which are useful for
long term expression by the vectors of the invention are described,
for example, in European Publication No. 0437478B1, entitled
"Cyclodextrin-Peptide Complexes." Nucleic acid sequences encoding a
variety of hormones can be used, including those encoding human
growth hormone, insulin, calcitonin, prolactin, follicle
stimulating hormone (FSH), luteinizing hormone (LH), human
chorionic gonadotropin (HCG), and thyroid stimulating hormone
(TSH). A variety of different forms of IGF-1 and IGF-2 growth
factor polypeptides are also well known the art and can be
incorporated into gene transfer vectors for long term expression in
vivo. See, e.g., European Patent No. 0123228B1, published for grant
Sep. 19, 1993, entitled "Hybrid DNA Synthesis of Mature
Insulin-like Growth Factors." As an additional example, the long
term in vivo expression of different forms of fibroblast growth
factor can also be effected by the methods of invention. See, e.g.,
U.S. Pat. Nos. 5,464,774, 5,155,214, and 4,994,559 for a
description of different fibroblast growth factors.
[0075] In particular embodiments, the gene transfer vectors of the
present invention may include a suicide gene and an ancillary
nucleotide sequence which can be expressed to provide immune
accessory molecules. As utilized herein, the phrase "immune
accessory molecules" refers to molecules which can either increase
or decrease the recognition, presentation or activation of an
immune response (either cell-mediated or humoral). Representative
examples of immune accessory molecules are described above.
[0076] When the gene transfer vectors described herein direct the
expression of more than one heterologous sequence, such multiple
sequences may be controlled either by a single promoter, or
preferably, by additional secondary promoters (e.g., Internal
Ribosome Binding Sites or "IRBS").
[0077] Polynucleotide sequences coding for the above-described
molecules can be obtained using recombinant methods, such as by
screening cDNA and genomic libraries from cells expressing the
gene, or by deriving the gene from a vector known to include the
same. For example, plasmids which contain sequences that encode
altered cellular products may be obtained from a depository such as
the ATCC, or from commercial sources such as Advanced
Biotechnologies (Columbia, Maryland). Plasmids containing the
nucleotide sequences of interest can be digested with appropriate
restriction enzymes, and DNA fragments containing the nucleotide
sequences can be inserted into a gene transfer vector using
standard molecular biology techniques.
[0078] Alternatively, cDNA sequences for use with the present
invention may be obtained from cells which express or contain the
sequences, using standard techniques, such as phenol extraction and
PCR of cDNA or genomic DNA. See, e.g., Sambrook et al., supra, for
a description of techniques used to obtain and isolate DNA.
Briefly, mRNA from a cell which expresses the gene of interest can
be reverse transcribed with reverse transcriptase using oligo-dT or
random primers. The single stranded cDNA may then be amplified by
PCR (see U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159, see
also PCR Technology: Principles and Applications for DNA
Amplification, Erlich (ed.), Stockton Press, 1989)) using
oligonucleotide primers complementary to sequences on either side
of desired sequences. In particular, a double stranded DNA is
denatured by heating in the presence of heat-stable Taq polymerase,
sequence-specific DNA primers, and ATP, CTP, GTP and TTP.
Double-stranded DNA is produced when synthesis is complete. This
cycle may be repeated many times, resulting in a factorial
amplification of the desired DNA.
[0079] The nucleotide sequence of interest can also be produced
synthetically, rather than cloned, using a DNA synthesizer (e.g.,
an Applied Biosystems Model 392 DNA Synthesizer, available from
ABI, Foster City, Calif.). The nucleotide sequence can be designed
with the appropriate codons for the expression product desired. In
general, one will select preferred codons for the intended host in
which the sequence will be expressed. The complete sequence is
assembled from overlapping oligonucleotides prepared by standard
methods and assembled into a complete coding sequence. See, e.g.,
Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299;
Jay et al. (1984) J. Biol. Chem. 259:6311.
[0080] Next, one or more coding sequences can be inserted into a
vector which includes control sequences operably linked to the
desired coding sequence(s), and which allow for in vivo expression
in the targeted host species. For example, typical promoters for
mammalian cell expression include the SV40 early promoter, a CMV
promoter such as the CMV immediate early promoter, the mouse
mammary tumor virus LTR promoter, the adenovirus major late
promoter (Ad MLP), and the herpes simplex virus promoter, among
others. Other nonviral promoters, such as a promoter derived from
the murine metallothionein gene, will also find use for mammalian
expression. Typically, transcription termination and
polyadenylation sequences will also be present, located 3' to the
translation stop codon. Preferably, a sequence for optimization of
initiation of translation, located 5' to the coding sequence, is
also present. Examples of transcription terminator/polyadenylation
signals include those derived from SV40, as described in Sambrook
et al., supra, as well as a bovine growth hormone terminator
sequence. Introns, containing splice donor and acceptor sites, may
also be designed into the constructs for use with the present
invention.
[0081] Enhancer elements may also be used herein to increase
expression levels of the vector constructs. Examples include the
SV40 early gene enhancer, as described in Dijkema et al. (1985)
EMBO J. 4:761, the enhancer/promoter derived from the long terminal
repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et
al. 91982) Proc. Natl. Acad. Sci. USA 79:6777 and elements derived
from human CMV, as described in Boshart et al. (1985) Cell 41:521,
such as elements included in the CMV intron A sequence.
[0082] A number of viral based systems have been developed for use
as gene transfer vectors for mammalian host cells. For example,
retroviruses provide a convenient platform for gene delivery
systems. A selected gene can be inserted into a vector and packaged
in retroviral particles using techniques known in the art. The
recombinant virus can then be isolated and delivered to cells of
the subject either in vivo or ex vivo. A number of retroviral
systems have been described and will find use with the present
invention, including, for example, those described in (U.S. Pat.
No. 5,219,740; Miller et al. (1989) BioTechniques 7:980; Miller,
A.D. (1990) Human Gene Therapy 1:5; Scarpa et al. (1991) Virology
180:849; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033;
Boris-Lawrie et al. (1993) Cur. Opin. Genet. Develop. 3:102; GB
2200651; EP 0415731; EP 0345242; WO 89/02468; WO 89/05349; WO
89/09271; WO 90/02806; WO 90/07936; WO 90/07936; WO 94/03622; WO
93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; in
U.S. Pat Nos. 5,219,740; 4,405,712; 4,861,719; 4,980,289 and
4,777,127; in U.S. Ser. No. 07/800,921; and in Vile (1993) Cancer
Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993)
Cancer Res 53:83-88; Takamiya (1992) J Neurosci Res 33:493-503;
Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane
(1984) Proc Natl Acad Sci USA 81;6349; and Miller (1990) Human Gene
Therapy 1. Retroviral gene transfer vectors are preferred in the
practice of the invention.
[0083] Retroviral gene transfer vectors used in the practice of the
present invention may be readily constructed from a wide variety of
retroviruses, including for example, B, C, and D type retroviruses
as well as spumaviruses and lentiviruses (see, e.g., RNA Tumor
Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
Briefly, retroviruses have been classified according to their
morphology as seen under electron microscopy. Type "B" retroviruses
appear to have an eccentric core, while type "C" retroviruses have
a central core. Type "D" retroviruses have a morphology
intermediate between type B and type C retroviruses. Representative
examples of suitable retroviruses include, for example, those
described in RNA Tumor Viruses, at pages 2-7, as well as a variety
of xenotropic retroviruses (e.g., NZB-X1, NZB-X2 and NZB9-1 (see
O'Neill et al. (1985) J. Vir. 53:100-106)) and polytropic
retroviruses (e.g., MCF and MCF-MLV (see Kelly et al. (1983) J.
Vir. 45(l):291-298)). Such retroviruses may be readily obtained
from depositories or collections such as the American Type Culture
Collection ("ATCC"; Rockville, Md.), or isolated from known sources
using commonly available techniques.
[0084] Particularly preferred retroviruses for the preparation or
construction of retroviral gene transfer vectors of the present
invention include retroviruses selected from the group consisting
of Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia
Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Gibbon
Ape Leukemia Virus, Feline Leukemia Virus, Reticuloendotheliosis
virus and Rous Sarcoma Virus. Particularly preferred Murine
Leukemia Viruses include 4070A and 1504A (Hartley et al. (1976) J.
Virol. 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No.
VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma
Virus and Rauscher (ATCC No. VR-998), and Moloney Murine Leukemia
Virus (ATCC No. VR-190). Particularly preferred Rous Sarcoma
Viruses include Bratislava, Bryan high titer (e.g., ATCC Nos.
VR-334, VR-657, VR-726, VR-659, and VR-728), Bryan standard,
Carr-Zilber, Engelbreth-Holm, Harris, Prague (e.g., ATCC Nos.
VR-772, and 45033), and Schmidt-Ruppin (e.g., ATCC Nos. VR-724,
VR-725, VR-354).
[0085] Any of the above retroviruses may be readily utilized in
order to assemble or construct retroviral gene transfer vectors
given the disclosure provided herein, and standard recombinant
techniques (e.g., Sambrook et al., supra; Kunkle (1985) Proc. Natl.
Acad. Sci. USA 82:488). Within certain embodiments of the
invention, portions of the retroviral gene transfer vectors may be
derived from different retroviruses. For example, retroviral vector
LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding
site from a Rous Sarcoma Virus, a packaging signal from a Murine
Leukemia Virus, and an origin of second strand synthesis from an
Avian Leukosis Virus.
[0086] Retroviral vector constructs can also be provided comprising
a 5' LTR, a tRNA binding site, a packaging signal, one or more
heterologous sequences, an origin of second strand DNA synthesis
and a 3' LTR, wherein the vector construct lacks gag/pol or env
coding sequences. Briefly, Long Terminal Repeats ("LTRs") are
subdivided into three elements, designated U5, R and U3. These
elements contain a variety of signals which are responsible for the
biological activity of a retrovirus, including for example,
promoter and enhancer elements which are located within U3. LTRs
may be readily identified in the provirus due to their precise
duplication at either end of the genome. As utilized herein, a 5'
LTR is understood to include a 5' promoter element and sufficient
LTR sequence to allow reverse transcription and integration of the
DNA form of the vector. The 3' LTR includes a polyadenylation
signal, and sufficient LTR sequence to allow reverse transcription
and integration of the DNA form of the vector.
[0087] The tRNA binding site and origin of second strand DNA
synthesis are also important for a retrovirus to be biologically
active, and may be readily identified by one of skill in the art.
For example, retroviral tRNA binds to a tRNA binding site by
Watson-Crick base pairing, and is carried with the retrovirus
genome into a viral particle. The tRNA is then utilized as a primer
for DNA synthesis by reverse transcriptase. The tRNA binding site
may be readily identified based upon its location immediately
downstream from the 5' LTR. Similarly, the origin of second strand
DNA synthesis is important for the second strand DNA synthesis of a
retrovirus. This region, which is also referred to as the
poly-purine tract, is located immediately upstream of the 3'
LTR.
[0088] In addition to a 5' and 3' LTR, tRNA binding site, and
origin of second strand DNA synthesis, the retroviral gene transfer
vectors may further comprise a packaging signal, as well as one or
more heterologous sequences, each of which is discussed in more
detail below.
[0089] For example, retroviral gene transfer vectors can be
provided which lack both gag/pol and env coding sequences. As an
illustration, construction of retroviral gene transfer vectors
which lack gag/pol or env sequences may be accomplished by
preparing vector constructs which lack an extended packaging
signal. As utilized herein, the phrase "extended packaging signal"
refers to a sequence of nucleotides beyond the minimum core
sequence which is required for packaging. The sequence allows
increased viral titer due to enhanced packaging. As an example, for
the Murine Leukemia Virus MoMLV, the minimum core packaging signal
is encoded by the sequence beginning from the end of the 5' LTR
through the PstI site. The extended packaging signal of MoMLV
includes the sequence beyond nucleotide 567 through the start of
the gag/pol gene (nucleotide 621), and beyond nucleotide 1560.
Thus, retroviral gene transfer vectors which lack extended
packaging signal may be constructed from the MoMLV by deleting or
truncating the packaging signal prior to nucleotide 567.
[0090] Other retroviral gene transfer vectors can be provided
wherein the packaging signal that extends into, or overlaps with,
retroviral gag/pol sequence is deleted or truncated. For example,
in the representative case of MoMLV, the packaging signal is
deleted or truncated prior to the start of the gag/pol gene.
[0091] Retroviral gene transfer vectors can also be provided to
include a packaging signal that extends beyond the start of the
gag/pol gene. When such retroviral vector constructs are utilized,
it is preferable to use packaging cell lines for the production of
recombinant viral particles wherein the 5' terminal end of the
gag/pol gene in a gag/pol expression cassette has been modified to
contain codons which are degenerate for gag.
[0092] Yet further retroviral vector constructs can be provided
which comprise a 5' LTR, a tRNA binding site, a packaging signal,
an origin of second strand DNA synthesis and a 3' LTR, wherein the
vector construct does not contain a retroviral nucleic acid
sequence upstream of the 5' LTR: These vector constructs do not
contain a env coding sequence upstream of the 5' LTR.
[0093] Retroviral gene transfer vectors can also be provided which
comprise a 5' LTR, a tRNA binding site, a packaging signal, an
origin of second strand DNA synthesis and a 3' LTR, wherein the
vector does not contain a retroviral packaging signal sequence
downstream of the 3' LTR. As utilized herein, the term "packaging
signal sequence" is understood to mean a sequence sufficient to
allow packaging of the RNA genome.
[0094] Packaging cell lines suitable for use with the above
described retroviral gene transfer vector constructs may be readily
prepared (see U.S. application Ser. No. 08/240,030, filed May 9,
1994; see also U.S. application Ser. No. 07/800,921, filed Nov. 27,
1991), and utilized to create producer cell lines (also termed
vector cell lines or "VCLs") for the production of recombinant
vector particles.
[0095] A number of viral vector systems other than those based on
retroviruses are known in the art and can be used in the practice
of the invention. Since the viral vector systems are used to
provide therapeutically useful modified cells, the viral vector
systems are preferably genetically modified to render them
non-lytic.
[0096] A number of adenovirus vectors (Ad vectors) have been
described and can be used with the present invention. See, e.g.,
Haj-Ahmad et al. (1986) J. Virol. 57:267; Bett et al. (1993) J.
Virol. 67:5911; Mittereder et al. (1994) Human Gene Therapy 5:717;
Seth et al. (1994) J. Virol. 68:933; Barr et al. (1994) Gene
Therapy 1:51; Berkner, K. L. (1988) BioTechniques 6:616; and Rich
et al. (1993) Human Gene Therapy 4:461.
[0097] Prototype recombinant adenovirus vectors are generally
deleted in the early region one (Ela/Elb, or El) region, rendering
them replication-defective. Following insertion of a nucleotide
sequence of interest into the deleted region, propagation of the
recombinant E1-deleted adenovirus vector is accomplished in 293
cells, a complementing human embryonic kidney cell line stably
transformed with the Ad E1 region, which provides the Ad E1 region
gene products in trans. Recombinant Ad vectors generated in this
fashion can yield preparations with titers between 10.sup.11 to
10.sup.13 particles/ml (reviewed in Berkner (1988) BioTechniques
6:616-629). However, there are several drawbacks to this prototype
Ad vector system, including: (1) size restriction of heterologous
genetic material to approximately 4.5 to 5.0 kb or less; and (2)
partial replication competence of the E1-deleted Ad vectors (Rich
(1993) Hum. Gen. Ther. 4:461-476). This later point arises in part
to a complementing "E1-like activity" that is expressed in human
cells, and results in the expression of other viral gene products
present in these vectors, including the highly immunogenic, "late,"
or structural gene products (e.g. penton protein). As a result of
immune responses of the recipient to Ad-specific proteins expressed
by the E1-deleted vectors, expression of the heterologous gene, or
transgene can be transient and associated with the development of
pathology at the site of gene transfer.
[0098] Thus, second generation Ad vectors have sought to further
"cripple" the capacity of the vector to replicate and express
viral-specific gene products, and to increase the capacity of
heterologous genetic material. Such vectors have been of three
types: (1) E1 and E3 genes deleted (Bett (1994) Proc. Natl. Acad.
Sci. USA 91:8802-8806); (2) E1 and E4 genes deleted (Wang (1995)
Gene Ther. 2:775-783); and (3) deletion of all Ad viral genes, or
"gutless" (Fisher (1996) Virology 217:11-22, Hardy (1996) J. Virol.
71:1842-1849, and Kochanek (1996) Proc. Natl. Acad. Sci. USA
93:5731-5736). The duration of transgene expression in animals
inoculated with these second generation recombinant adenovirus
vectors has been dramatically increased as a result of the
mitigation of the recipient's immune response to the Ad
vectors.
[0099] As expected, increased deletion of viral-specific genes in
the second generation Ad vectors has also resulted in an increased
capacity for heterologous genetic material, thus extending the
usefulness of this system for application to human gene transfer.
This capacity for heterologous genetic material is approximately 8
kb in the E1/E3 and E1/E4 vectors, and is greater than 30 kb for
the "gutless" Ad vectors, permitting the insertion of entire genes,
including relevant gene expression control regions.
[0100] Generation of recombinant Ad vectors, including the E1/E3,
E1/E4, and "gutless" vectors, can be accomplished according to
methods well-known to those skilled in the art. For example: (1)
nucleotide sequences of interest can be inserted into plasmid pBHG1
(Bett (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806), to generate
recombinant E1/E3-deleted Ad vectors after transfection of 293
cells and subsequent intracellular homologous recombination; (2)
nucleotide sequences of interest can be first substituted into the
E1 region of any of a variety of E1-deleted Ad vectors and
co-transfected with ClaI digested H5dl1014, and recombinant
E1/E4-deleted Ad vectors generated after transfection of 293-E4
cells (Wang (1995) Gene Ther. 2:775-783), and subsequent
intracellular homologous recombination; and (3) the nucleotide
sequences of interest can first be inserted into the .DELTA.rAd
plasmid (Fisher (1996) Virology 217:11-22), along with appropriate
amounts of "stuffer" sequence derived from, for example,
bacteriophage lambda DNA, to permit efficient packaging of
recombinant "gutless" adenovirus vector genomes, transfected onto
293 cells and infected with H5.CBALP helper virus (Yang (1995)
Virology 69:2004-2015). Purification of recombinant "gutless"
adenovirus vector particles from helper virus can be accomplished,
for example, by centrifugation over a cesium gradient, as a result
of a buoyant density lower than that of helper virus.
[0101] Additionally, various adeno-associated virus (AAV) vector
systems have been developed for gene delivery and find use herein.
AAV vectors can be readily constructed using techniques well known
in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941;
International Publication Nos. WO 92/01070 (published 23 January
1992) and WO 93/03769 (published Mar. 4, 1993); commonly owned
provisional U.S. patent application Ser. No. 60/025649; Lebkowski
et al. (1988) Molec. Cell. Biol. 8:3988; Vincent et al. (1990)
Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J.
(1992) Current Opinion in Biotechnology 3:533; Muzyczka, N. (1992)
Current Topics in Microbiol. and Immunol. 158:97; Kotin, R. M.
(1994) Human Gene Therapy 5:793; Shelling et al. (1994) Gene
Therapy 1:165; and Zhou et al. (1994) J. Exp. Med. 179:1867.
[0102] Additional viral vectors which will find use for delivering
the nucleic acid molecules encoding the nucleotide sequences of
interest include those derived from the pox family of viruses,
including vaccinia virus and avian poxvirus. By way of example,
vaccinia virus recombinants expressing the genes can be constructed
as follows. DNA encoding the particular gene is first inserted into
an appropriate vector so that it is adjacent to a vaccinia promoter
and flanking vaccinia DNA sequences, such as the sequence encoding
thymidine kinase (TK). This vector is then used to transfect cells
which are simultaneously infected with vaccinia. Homologous
recombination serves to insert the vaccinia promoter plus the gene
into the viral genome. The resulting TK.sup.- recombinant can be
selected by culturing the cells in the presence of
5-bromodeoxyuridine and picking viral plaques resistant
thereto.
[0103] Alternatively, avipoxviruses, such as the fowlpox and
canarypox viruses, can also be used to deliver the genes.
Recombinant avipox viruses, expressing immunogens from mammalian
pathogens, are known to confer protective immunity when
administered to non-avian species. The use of an avipox vector is
particularly desirable in human and other mammalian species since
members of the avipox genus can only productively replicate in
susceptible avian species and therefore are not infective in
mammalian cells. Methods for producing recombinant avipoxviruses
are known in the art and employ genetic recombination, as described
above with respect to the production of vaccinia viruses. See,
e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
[0104] Molecular conjugate vectors, such as the adenovirus chimeric
vectors described in Michael et al. (1993) J. Biol. Chem. 268:6866
and Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:6099, can
also be used for gene delivery.
[0105] Members of the Alphavirus genus, such as but not limited to
vectors derived from the Sindbis and Semliki Forest viruses, will
also find use as gene delivery vectors for delivering a nucleotide
sequence of interest. For a description of Sinbus-virus derived
vectors useful for the practice of the instant methods. See, e.g.,
Dubensky et al. (1996) J. Virol. 70:508; and International
Publication Nos. WO 95/07995 and WO 96/17072.
[0106] A number of non-viral based gene delivery systems have also
been developed for use as gene transfer vectors for mammalian host
cells including, for example, nucleic acid expression vectors;
polycationic condensed DNA linked or unlinked to killed adenovirus
alone (see e.g., U.S. patent application Ser. No. 08/366,787, filed
Dec. 30, 1994, and Curiel (1992) Hum. Gene Ther. 3:147-154); ligand
linked DNA (see Wu (1989) J. Biol. Chem. 264:16985-16987);
eukaryotic cell delivery vehicles cells (see U.S. patent
application Ser. No.08/240,030, filed May 9, 1994, and U.S. patent
application Ser. No. 08/404,796); deposition of photopolymerized
hydrogel materials; hand-held gene transfer particle gun (see,
e.g., U.S. Pat. No. 5,149,655); ionizing radiation (e.g., as
described in U.S. Pat. No. 5,206,152 and in International
Publication No. WO 92/11033); nucleic charge neutralization or
fusion with cell membranes. Additional approaches are described in
Philip (1994) Mol. Cell Biol. 14:2411-2418, and in Woffendin (1994)
Proc. Natl. Acad. Sci. USA 91:1581-1585.
[0107] Particle mediated gene transfer may be employed with
non-viral based systems, for example, see U.S. provisional
application No. 60/023,867. Briefly, the sequence of interest can
be inserted into conventional gene transfer vectors containing
suitable control sequences for high level expression, and then be
incubated with synthetic gene transfer molecules such as polymeric
DNA-binding cations like polylysine, protamine, and albumin, linked
to cell targeting ligands such as asialoorosomucoid (e.g., as
described in Wu et al. (1987) J. Biol. Chem. 262:4429-4432),
insulin (e.g., as described in Hucked (1990) Biochem. Pharmacol.
40:253-263), galactose (e.g., as described in Plank (1992)
Bioconjugate Chem. 3:533-539), lactose or transferrin.
[0108] Naked DNA delivery techniques may also be employed.
Exemplary naked DNA introduction methods are described in WO
90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be
improved using biodegradable latex beads. DNA coated latex beads
are efficiently transported into cells after endocytosis initiation
by the beads. The method may be improved further by treatment of
the beads to increase hydrophobicity and thereby facilitate
disruption of the endosome and release of the DNA into the
cytoplasm.
[0109] Liposomes that act as vehicles for gene transfer vectors are
described in U.S. Pat. No. 5,422,120, International Publication
Nos. WO 95/13796, WO 94/23697, and WO 91/144445, and in European
Patent Publication No. 524,968. As described in U.S. provisional
application No. 60/023,867, nucleic acid sequences can be inserted
into vectors having control sequences suitable for high level
expression, and then incubated with synthetic gene transfer
molecules such as polymeric DNA-binding cations like polylysine,
protamine, and albumin, linked to cell targeting ligands such as
asialoorosomucoid, insulin, galactose, lactose, or transferrin.
Other delivery systems include the use of liposomes to encapsulate
DNA comprising the gene under the control of a variety of
tissue-specific or ubiquitously-active promoters. Further non-viral
delivery techniques suitable for use herein include mechanical
delivery systems such as the approach described in Woffendin et al.
(1994) Proc. Natl. Acad. Sci. USA 91:11581-11585. Moreover, the
coding sequence can be delivered through deposition of
photopolymerized hydrogel materials. Other conventional methods for
gene delivery that can be used include, for example, use of
hand-held gene transfer particle gun, as described in U.S. Pat. No.
5,149,655; and ionizing radiation for activating transferred gene,
as described in U.S. Pat. No. 5,206,152 and International
Publication No. WO 92/11033.
[0110] Exemplary liposome and polycationic gene delivery vehicles
are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915, in
International Publication Nos. WO 95/13796, WO 94/23697, and WO
91/14445, in European Patent Publication No. 524,968 and in
Starrier, Biochemistry, pp 236-240 (1975) W. H. Freeman, San
Francisco; Shokai (1980) Biochem. Biophys. Acct. 600:1; Bayer
(1979) Biochem. Biophys. Acct. 550:464; Rivet (1987) Meth. Enzymol.
149:119; Wang (1987) Proc. Natl. Acad. Sci. USA 84:7851; Plant
(1989) Anal. Biochem. 176:420.
[0111] Once produced, the above-described gene transfer vectors are
used to genetically modify a population of T cells isolated as
described herein. If a retroviral gene transfer vector is used, a
population of cultured T cells is first activated by stimulating
the cells to the part of S-phase which is most receptive to
transfection. The first quarter to half of S-phase is optimal for
retroviral transduction. Other methods of enhancing a cell's
receptivity to viral vector transduction include varying the
multiplicity of infection, depleting ions such as phosphate, adding
polycations such as protamine sulfate, adjusting the contact time,
temperature, pH, and centrifuging the cells and viruses
together.
[0112] In the practice of the invention, T-lymphocytes are
preferably activated by contacting them with a CD3-binding agent
such as the monoclonal antibody OKT-3. A CD3-binding agent is a
ligand which binds to the CD3 molecule on the surface of cells. The
ligand can be an antibody, such as OKT-3, which can cross-link two
or more CD3 molecules. Such cross-linking can be responsible for
the proliferation and activation of CD3-bearing cells such as
T-lymphocytes. The activation of T-lymphocytes by CD3-binding
agents is increased by adjusting certain factors such as binding
agent concentration, time of contact, number of cells, temperature
of contact, and the binding agent's affinity, avidity, and efficacy
of activating the cells.
[0113] The T cells can also be maintained in a medium containing at
least one type of growth factor prior to being selected. A variety
of growth factors are known in the art which sustain the growth of
a particular cell type. Examples of such growth factors are
cytokine mitogens such as rIL-2, IL-10, IL-12, and IL-15, which
promote growth and activation of lymphocytes. Certain types of
cells are stimulated by other growth factors such as hormones,
including human chorionic gonadotropin (hCG) and human growth
hormone. The selection of an appropriate growth factor for a
particular cell population is readily accomplished by one of skill
in the art.
[0114] For example, white blood cells such as differentiated
progenitor and stem cells are stimulated by a variety of growth
factors. More particularly, IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF,
M-CSF, and G-CSF, produced by activated T.sub.H and activated
macrophages, stimulate myeloid stem cells, which then differentiate
into pluripotent stem cells, granulocyte-monocyte progenitors,
eosinophil progenitors, basophil progenitors, megakaryocytes, and
erythroid progenitors. Differentiation is modulated by growth
factors such as GM-CSF, IL-3, IL-6, IL-11, and EPO.
[0115] Pluripotent stem cells then differentiate into lymphoid stem
cells, bone marrow stromal cells, T cell progenitors, B cell
progenitors, thymocytes, T.sub.H Cells, T.sub.c cells, and B cells.
This differentiation is modulated by growth factors such as IL-3,
IL-4, IL-6, IL-7, GM-CSF, M-CSF, G-CSF, IL-2, and IL-5.
[0116] Granulocyte-monocyte progenitors differentiate to monocytes,
macrophages, and neutrophils. Such differentiation is modulated by
the growth factors GM-CSF, M-CSF, and IL-8. Eosinophil progenitors
differentiate into eosinophils. This process is modulated by GM-CSF
and IL-5.
[0117] The differentiation of basophil progenitors into mast cells
and basophils is modulated by GM-CSF, IL-4, and IL-9.
Megakaryocytes produce platelets in response to GM-CSF, EPO, and
IL-6. Erythroid progenitor cells differentiate into red blood cells
in response to EPO.
[0118] Thus, during activation by the CD3-binding agent, T cells
can also be contacted with a mitogen, for example a cytokine such
as IL-2. In particularly preferred embodiments, the IL-2 is added
to the population of T cells at a concentration of about 50 to 100
.mu.g/ml. Activation with the CD3-binding agent can be carried out
for 2 to 4 days.
[0119] Once suitably activated, the T cells are genetically
modified by contacting the same with a suitable gene transfer
vector under conditions that allow for transfection of the vectors
into the T cells. Genetic modification is carried out when the cell
density of the T cell population is between about
0.1.times.10.sup.6 and 5.times.10.sup.6, preferably between about
0.5.times.10.sup.6 and 2.times.10.sup.6. Although a number of
suitable viral and nonviral-based gene transfer vectors have been
described for use herein, the invention is hereafter exemplified by
transduction of the T cells using a viral-based vector system.
[0120] Transduction with a gene transfer vector is generally
carried out with a viral vector at a multiplicity of infection
(MOI) of about 3 or greater.
[0121] In one embodiment, the T cells are washed after activation
with the CD3-binding agent, and then re-seeded in cell culture at a
cell density of about 5.times.10.sup.5.
[0122] In another particular embodiment, the gene transfer vector
contains a promoter operably linked to a first nucleotide sequence
that is capable of being expressed in a transduced cell to provide
the cell with enhanced susceptibility to a selected cytotoxic
agent. Preferably, the first nucleotide sequence is a suicide gene,
such as the herpes simplex virus thymidine kinase (HSV-tk) gene.
The gene transfer vector can also include a selectable marker. A
number of suitable selectable markers can be used in the practice
of the invention, such as those which provide a transduced cell
with resistance to a selected cytotoxic agent. One particular
selectable marker for use herein is neomycin phosphotransferase II.
Other markers useful herein include cell surface markers such as
alkaline phosphatase, nerve growth factor, or any other suitable
membrane-associated moiety.
[0123] The gene transfer vector used in this embodiment of the
invention is preferably a retroviral vector, containing a suicide
gene and a suitable selectable marker. The retroviruses used in the
Examples which follow are the product of the following vectors: 1)
pLXSN-T84.66g, 2) pLXSN-N29g, and the Tk retroviral vector which
produces the DAHSVTK9A retrovirus (Viagene, San Diego, Calif.). The
maps of the first two vectors are provided in FIGS. 1 and 2,
respectively. The overall map of the Tk retroviral vector
(DAHSVTK9A) is shown in FIG. 3. FIG. 4 depicts the structure of the
RVV HSV-TK provector.
[0124] The pLXSN-T84.66g and pLXSN-N29g vectors have the neo gene
under control of the SV40 promoter, and retroviral 5' LTR, 3' LTR
and .PSI. packaging sequences. The retroviral Tk vector (DAHSVTK9A)
has a HSV-tk gene transcribed under control of the Moloney 5' LTR
early promoter and a neo gene transcribed under control of the SV40
promoter. The RVV HSV-TK provector also has a HSV-tk gene
transcribed under control of the Moloney 5' LTR early promoter and
a neo gene transcribed under control of the SV40 promoter.
[0125] Thus, in one embodiment, a retroviral gene delivery vector
including a suicide gene (e.g., a HSV-tk gene) is prepared as
described above, and used to transduce a population of T cells as
described above. General T cell transduction methodologies are
described in commonly owned U.S. patent application Ser. No.
08/425,180, entitled "High Efficiency ex vivo Transduction of Cells
by High Titer Recombinant Retroviral Preparations," which
application is incorporated herein by reference. Other methods of
growing and transducing T cells can be used and are known to those
skilled in the art (e.g., Chuck et al. (1996) Hum. Gene Ther.
7:743; Heslop et al. (1996) Nature Med. 2:551; Riddell et al.
(1996) Nature Medicine 2:216). T cells can also be transduced by
methods used to grow and transduce T cells from HIV patients (e.g.,
Vandenddriessche et al. (1995) J. Virol. 69:4045; Sun et al. (1995)
Proc. Natl. Acad. Sci. USA 92:7272).
[0126] The T cell-transduction method described herein can be used
to obtain a transduction efficiency of 100% or greater in a
non-selected population of transduced T cells. This transduction
efficiency has heretofore not been attainable using prior
methodology.
[0127] After transduction, transduced cells are selected away from
non-transduced cells using known techniques. For example, if the
gene transfer vector used in the transduction includes a selectable
marker which confers resistance to a cytotoxic agent, the cells can
be contacted with the appropriate cytotoxic agent, whereby
non-transduced cells can be negatively selected away from the
transduced cells. If the selectable marker is a cell surface
marker, the cells can be contacted with a binding agent specific
for the particular cell surface marker, whereby the transduced
cells can be positively selected away from the population. The
selection step can also entail fluorescence-activated cell sorting
(FACS) techniques, such as where FACS is used to select cells from
the population containing a particular surface marker, or the
selection step can entail the use of magnetically responsive
particles as retrievable supports for target cell capture and/or
background removal.
[0128] More particularly, positive selection of the transduced
cells can be performed using a FACS cell sorter (e.g. a
FACSVantage.TM. Cell Sorter, Becton Dickinson Immunocytometry
Systems, San Jose, Calif.) to sort and collect transduced cells
expressing a selectable cell surface marker. Following
transduction, the cells are stained with fluorescent-labeled
antibody molecules directed against the particular cell surface
marker. The amount of bound antibody on each cell can be measured
by passing droplets containing the cells through the cell sorter.
By imparting an electromagnetic charge to droplets containing the
stained cells, the transduced cells can be separated from other
cells. The positively selected cells are then harvested in sterile
collection vessels. These cell sorting procedures are described in
detail, for example, in the FACSVantage.TM. Training Manual, with
particular reference to sections 3-11 to 3-28 and 10-1 to
10-17.
[0129] Positive selection of the transduced cells can also be
performed using magnetic separation of cells based on expression or
a particular cell surface marker. IN such separation techniques,
cells to be positively selected are first contacted with specific
binding agent (e.g., an antibody or reagent the interacts
specifically with the cell surface marker). The cells are then
contacted with retrievable particles (e.g., magnetically responsive
particles) which are coupled with a reagent that binds the specific
binding agent (that has bound to the positive cells). The
cell-binding agent-particle complex can then be physically
separated from non-labelled cells, for example using a magnetic
field. When using magnetically responsive particles, the labelled
cells can be retained in a container using a magnetic filed while
the negative cells are removed. These and similar separation
procedures are described, for example, in the Baxter Immunotherapy
Isolex training manual.
[0130] Expression of the vector in the selected transduced cells
can be assessed by a number of assays known to those skilled in the
art. For example, Western blot or Northern analysis can be employed
depending on the nature of the inserted nucleotide sequence of
interest. Once expression has been established and the transformed
T cells have been tested for the presence of adventitious agents,
they are ready for infusion into a patient via the peripheral blood
stream.
[0131] The invention further includes a kit for genetic
modification of an ex vivo population of primary mammalian cells.
The kit contains a gene transfer vector coding for at least one
selectable marker and at least one nucleotide sequence of interest
contained in one or more containers, ancillary reagents or
hardware, and instructions for use of the kit. The instructions can
be recorded on any suitable medium such as paper, plastic, magnetic
media, or on a CD.
[0132] The container or containers can be hermetically sealed so as
to physically separate the contents from the environment to prevent
the exchange of moisture, gases, particles, microbes, viruses, and
the like. Such containers can be glass or plastic vials, ampules,
or rubber-stopped containers from which samples can be repeatedly
removed using, for example, a syringe. The gene transfer vector
therein can be stored in a frozen, liquid, or lyophilized form in a
variety of media designed for the storing of such vectors. Storage
conditions such as temperature, time, and storage media, will vary
depending on the particular vector used, and are readily determined
by one of skill in the art. The containers can also include other
reagents such as buffers (i.e. PBS), salts (i.e. multivalent ions),
and stabilizing and preserving agents (i.e. glycerol and
antioxidants). Additional reagents useful for practicing the
particular genetic modification method can be included in the vial
containing the gene transfer vector or in other vials.
[0133] Ancillary reagents and/or hardware can also be contained in
the kits. Examples are buffers, reagents, containers, syringes,
pipettes, needles, tubing, biocompatible plastic bags, closed fluid
pathways, closed culture environments, and the like. All ancillary
reagents and hardware necessary to practice a method need not be
provided in a single kit.
[0134] In one particular embodiment, a production kit is provided.
The production kit contains and/or describes all components,
elements and processes necessary for ex vivo production of
genetically modified primary mammalian cells. The components of the
production kit can thus comprise or describe: (1) devices and
hardware systems (e.g., cell separation and/or processing equipment
for production of transduced cells, for example, a Fenwal.RTM.
Model CS3000 blood cell separator, a Terumo Sterile Connect Device
(Baxter), and the like); (2) a gene within a gene delivery vector
(the gene of interest contained within a suitable gene delivery
vector, wherein the gene delivery vector is suitably formulated for
inclusion within a sterile closed single-use container configured
for use with the above-described devices and hardware systems); (3)
disposable containers (e.g., disposable single-use containers for
manipulation, culture, handling, and/or cryopreservation of the ex
vivo modified cells, wherein the containers are sterile,
biocompatable, and suitable for use with closed fluid pathway
maintenance and configured for use with the above-described devices
and hardware systems); (4) reagents and solutions (e.g., any
reagents and/or solutions for use in the manipulation, culture,
handling or cryopreservation of the ex vivo modified cells, wherein
the reagents and solutions can be contained within sterile
containers and adapted for use with the above-described devices and
hardware systems); (5) biologics (e.g., biological agents and/or
reagents for use in the manipulation, culture, handling or
cryopreservation of the ex vivo modified cells, including growth
factors, mitogens, and cell selection reagents such as antibody
molecules or other specific binding agents); (6) ancillary reagents
(e.g., those used for selection and/or enrichment of cells
expressing selectable markers from a population of genetically
modified cells, such as magnetically responsive particles or
selection reagents (G418)); and (7) instructions (e.g., protocol
describing the manufacturing procedures required to produce
genetically modified cells in accordance with the present
invention).
[0135] Thus, a number of embodiments of the invention have been
described. In the Examples below, particular embodiments of the
invention are exemplified. In Example 1, high efficiency
transduction methods are used to produce 1.times.10.sup.9
Tk-Neo.sup.R-transduced T cells. This cell number will accommodate
both multiple dosing in an ex vivo gene therapy protocol and all
appropriate quality control sampling. To date, most clinical
studies have been performed in an open culture system using
multi-well tissue culture plates. These systems are not acceptable
or practical for generating clinical materials because they are
susceptible to contamination and are not commercially useful. In
the methods of the following examples, automated washing procedures
allow for complete medium exchanges whereby ancillary components
such as bovine serum are removed from the culture media in volume
exchange procedures.
[0136] High-dose chemotherapy followed by allogeneic bone marrow
transplantation (BMT) for the treatment of multiple myeloma and
leukemia has a curative potential attributed to graft vs. host
disease (GVHD) and graft vs. leukemia (GVL) effects. However,
allogeneic BMT recipients have an unreasonably high incidence of
severe and lethal GVHD. To solve this problem, T cell depletion of
the BMT has been used. Using this approach, the incidence of GVHD
is decreased; However, engraftment success and patient survival has
also decreased. Therefore, a preferred strategy in allogeneic BMT
would combine a means of effectively controlling GVHD without
interfering with GVL or engraftment.
[0137] One strategy, then, is to genetically modify donor T cells
to express a suicide gene prior to infusion. For example, the
methods of the invention can be used to genetically modify T cells
to express the Herpes Simplex Virus Thymidine Kinase (HSV-TK)
suicide gene. Such genetically modified T cells (expressing HSV-TK)
are thus rendered susceptible to ganciclovir (GCV) (Syntex
Laboratories, Palo Alto, Calif.), a drug tolerated by unmodified
cells but deleterious to cells which express HSV-TK. Because the
initial activation of the prodrug is catalyzed by viral thymidine
kinase, only the genetically modified cells are affected. These
modified donor T cells can then be infused with T cell-depleted
bone marrow to provide the beneficial effects of GVL with
engraftment. Subsequent GVHD can be controlled by administering GCV
to reduce alloreactive T cells.
[0138] The generation of up to about 1.times.10.sup.11 genetically
modified T lymphocytes for adoptive cell transfer poses several
challenges. Manual large-scale production techniques for generating
lymphokine-activated killer cells (LAK) for tumor immunotherapy
requires manual separation, washing, and centrifugation. The
processing of cells for each patient requires approximately 400
entries into tubes, flasks and roller bottles (Lee et al. (1994)
Transfusion Medicine 8:1203). The procedures described herein
provide several advantages in large-scale production methods by
eliminating a majority of the above-described manual techniques in
a closed fluid system that maximizes the use of automated fluid
separation and/or handling techniques.
[0139] After transduction, the T cells may be administered to a
suitable vertebrate subject. In addition, although warm-blooded
animals (e.g., mammals or vertebrates such as humans, macaques,
horses, cows, swine, sheep, dogs, cats, chickens, rats and mice)
have been exemplified in the methods described above, such methods
are also readily applicable to a variety of other animals,
including, for example, fish.
[0140] III. Experimental
[0141] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0142] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
EXAMPLE 1
High Efficiency Transduction of T Cells
[0143] This example demonstrates an efficient cell-processing
procedure in which T cells are activated, transduced with
Tk-Neo.sup.R retrovirus, selected in G418, and expanded in a closed
system. This procedure readily yields at least 1.times.10.sup.9
transduced T cells and demonstrates the feasibility of using this
protocol for the transduction of T cells on the scale needed for
clinical applications.
[0144] In order to evaluate the best media for activation of T
cells by OKT-3, the growth kinetics of T cells cultured in 31
different growth media were determined as follows. Briefly,
mononuclear cells were isolated from freshly drawn peripheral blood
using Ficoll-Hypaque.RTM. (Sigma Chemical Co., St. Louis, Mo.)
density-gradient separation. Peripheral blood mononuclear cells
were seeded in 6-well plates at 0.5.times.10.sup.6 cell/ml in each
medium or solution containing 60IU/ml rIL-2 (recombinant human
IL-2, Chiron, Emeryville, Calif.) and 10 ng/ml OKT-3 antibody
(Ortho Biotech, Raritan, N.J.), and cultured at 37.degree. C. in 5%
CO.sub.2. For each experiment, control mononuclear cells were
seeded at 0.5.times.10.sup.6 cells/ml in AIM V (GIBCO/BRL Life
Technologies, Gaithersberg, Md.)+10% Fetal Bovine Serum (FBS;
Biowhittaker, Walkersville, Md.), 60IU/ml rIL-2 and 10 ng/ml
OKT-3.
[0145] Three days after activation by OKT-3, cells were harvested
by centrifugation, counted, and resuspended in the same media or
solution without OKT-3 at 0.5.times.10.sup.6 cells/ml. Viability
was determined by trypan blue exclusion. Cell cycle analysis was
performed by FACS analysis before OKT-3-activation and three days
after OKT-3-activation. From these data, the percentage of cells in
the (S+G2/M) stage of the cell growth cycle was calculated for each
of the media tested. AIM V +7% FBS media resulted in the largest
percentage of cells in the S+G2/M stage, and was thus selected for
further development of transduction and cell processing protocols
as set forth below.
Large Scale Cell Processing and Transduction Protocol
[0146] T cells were isolated, OKT-3-activated, transduced,
G418-selected, and expanded as described below. T cells were
obtained from the leukapheresis product of three donors,
hereinafter referred to as A05, A06, and A07. For production of the
apheresis product, a suitable blood separator (e.g., a CS3000 Blood
Cell Separator (Baxter Fenwall) or a Cobe Spectra Hemometrics
separator) was used.
Isolation of Mononuclear Cells by Ficoll-Hypaque.RTM. Procedure
[0147] In order to provide purified human mononuclear cells, the
following procedure was carried out. The apheresis product of human
donors A05, A06 and A07 was isolated using Open System Apheresis
Kits (Baxter Fenwal) according to the manufacturer's instructions.
The apheresis product was transferred to Lifecell.RTM. Product
Flasks containing 200 ml of saline/ACD solution (Baxter Fenwal) and
diluted using the Fenwal.RTM. CS-3000.RTM. Plus Blood Cell
Separator (Baxter Fenwal) following the manufacturer's
instructions. The apheresis product had at least about
3.times.10.sup.9 mononuclear cells. Apheresis products having less
than about this number of cells were not taken any farther in the
procedure. The characteristics of the cells thus collected are set
forth in Table 1.
1TABLE 1 CELL PARAMETERS OF APHERESIS PRODUCTS Characteristic Donor
A05 Donor A06 Donor A07 Viability (%) 99 99 99 Vol. (ml) 161 172
172 Cell Density (.times.10.sup.6/ml) 25.4 22.4 35.7 Total#
(.times.10.sup.9/ml) 4.1 3.9 6.1 % Cells in S + G2/M 0.2 1.1 0.3 CD
Marker Profile % CD3+ 72 39 61 % CD3+ CD4+ 46 23 42 % CD3+ CD8+ 26
17 21 % CD3- CD16+ 11 26 16 % CD3+ CD56+ 1 5 6 % CD3- CD56+ 12 25
21 % CD3+ CD25+ 2 2 8 % CD3+ CD28+ 67 30 51 % CD3+ HLA-DR+ 2 2 2 %
CD3- HLA-DR+ 14 35 16
[0148] Mononuclear cells were isolated from diluted apheresis
product using the Fenwal.RTM. CS-3000.RTM. Plus by
Ficoll-Hypaque.RTM. density centrifugation following the
manufacturer's instructions. The Ficoll.RTM.-Hypaque purification
procedure yielded about 2.times.10.sup.9 to about 5.times.10.sup.9
mononuclear cells. The purified cellular product must have
approximately 1.times.10.sup.9 mononuclear cells in order to be
used in the later procedures. A portion of the mononuclear cells
(3.times.10.sup.8) was reserved for OKT-3 activation. Excess cells
were cryopreserved in Cryocyte.RTM. containers (Baxter Fenwal). The
yield and viability of the Ficoll.RTM.-Hypaque.RTM. purified cells
were determined; the results are shown in Table 2.
[0149] The Ficoll.RTM.-Hypaque.RTM. purified mononuclear cell
product was transferred using sterile technique from the
CS-3000.RTM. Plus Harvest Chamber to a tared Fenwal.RTM. Transfer
Pack Flask. Collection of quality control samples from closed
system Lifecell.RTM. Flasks was performed using a Fenwal.RTM.
Plasma Transfer Set (Baxter Fenwal). The contents of the
Lifecell.RTM. Flask were mixed to attain an even cell suspension
before a portion of the culture was drained from the Lifecell.RTM.
Flask into a sterile sample tube.
[0150] Cells were frozen in aliquots of no more than
50.times.10.sup.6 cells/ml in Cryocyte.RTM. containers with 1/3
cell volume of 3.times.Freezing Solution (10% AIM V media, 60% FBS,
and 30% DMSO). If desired, the freezing solution can employ 20%
autologous plasma in order to avoid bovine products (FBS). Frozen
cell samples were stored in liquid nitrogen.
2TABLE 2 CHARACTERISTICS OF FICOLL .RTM.-PURIFIED WHITE BLOOD CELLS
Characteristic DONOR A05 DONOR A06 DONOR A07 Viability % 95 93 96
Vol. (ml) 188 197 185 Cell Density (.times.10.sup.6/ml) 9.9 13.7
18.3 Total# (.times.10.sup.9/ml) 1.9 2.7 3.4 Recovery (%) 46 69
55
OKT-3 Activation of the T-Cells
[0151] In order to ensure that the cells were optimally receptive
to infection by retrovirus, the following activation procedure was
carried out. At least about 3.times.10.sup.8
Ficoll-Hypaque.RTM.-purified mononuclear cells were distributed
equally into 1L Lifecell.RTM. Flasks in Lymphocyte Activation Media
(AIM V with 10% FBS, 2 mM glutamine, 60IU/ml rIL-2 and 10 ng/ml
OKT-3) to achieve a final cell concentration of 5.times.10.sup.5
mononuclear cells/ml in about 200 to about 400 mL of media.
Ficoll.RTM.-purified mononuclear cell product first was manually
distributed into empty Lifecell.RTM. Flasks using Sepacell.RTM.
Adapter Sets and a Plasma Transfer Set, and a 60 cc syringe,
followed by the Activation Media.
[0152] The Lifecell.RTM. Flasks containing the Ficoll.RTM.-purified
cells in Lymphocyte Activation Media were incubated on wire racks
at about 37.degree. C. at about 5% CO.sub.2 for approximately three
days. Wire racks were used to enhance gas exchange. The results of
the OKT-3-activation procedures described above are summarized in
Table 3.
3TABLE 3 OKT-3-ACTIVATION OKT-3 Activation DONOR A05 DONOR A06
DONOR A07 # cells set up (.times.10.sup.8) 3 3 3 Viability 95 93 96
# cells/ml set up 0.5 0.5 0.5 (.times.10.sup.6) Total Volume (ml)
600 600 600 CD Marker Profile % CD3+ 47 24 53 % CD3+ CD4+ 33 15 38
% CD3+ CD8+ 14 10 16 % CD3- CD16+ 18 37 2 % CD3+ CD56+ 0 3 6 % CD3-
CD56+ 21 35 23 % CD3+ CD25+ 1 2 5 % CD3+ CD28+ 43 17 40 % CD3+
HLA-DR+ 2 2 2 % CD3- HLA-DR+ 36 38 24
Harvesting of T-cells Prior to Retroviral Transduction
[0153] About three days after OKT-3 activation, the activated
mononuclear cells were harvested from the Lymphocyte Activation
Media using the single chamber method of the CS-3000.RTM. Plus
essentially as set forth in the manufacturer's instructions.
Samples of the OKT-3-activated cells were taken aseptically for
quality control testing using the Tube Stripper (Baxter Fenwal) to
obtain a small length of tubing containing the aliquot to be
tested. The harvested activated mononuclear cells were transferred
to tared 1L Lifecell.RTM. Flasks, retaining 20 ml samples for
quality control testing.
[0154] The characteristics of the harvested activated mononuclear
cells are set forth below in Table 4. As shown in Table 4, the
activation procedure yielded greater than about 1.times.10.sup.8
mononuclear cells. The glucose concentration of the culture was
greater than about 100 mg/dL, and the lactate concentration was
less than about 1.0 mg/ml. These parameters are the threshold
release criteria for the methods which follow.
[0155] In preparation for transduction, at least about
1.5.times.10.sup.8 harvested, OKT-3-activated mononuclear cells in
Lymphocyte Transduction Media (AIM V with 10% FBS, 2 mM glutamine,
60IU/rIL-2, and 5 .mu.g/ml Protamine sulfate) were delivered
manually into 1L Lifecell.RTM. Flasks at a concentration of about
5.times.10.sup.5 mononuclear cells/ml. The preferred total volume
of the combined harvested, activated mononuclear cells and
Lymphocyte Transduction Media was about 200 to about 400 ml per 1
Liter Lifecell.RTM. Flask, with a maximum volume of about 500 ml.
Small samples (5 ml each) of the combined harvested, activated
mononuclear cells and the Lymphocyte Transduction Media were frozen
for later sterility testing at a later time. The concentration of
glucose and lactate in the samples was determined using a YSI-2700
Glucose Analyzer (Yellow Springs Instrument Co., Yellow Springs,
Ohio) following the manufacturer's instructions.
4TABLE 4 CHARACTERISTICS OF HARVESTED ACTIVATED MONONUCLEAR CELLS
DONOR A05 DONOR A06 DONOR A07 Single Single Single Chamber Chamber
Chamber on CS- on CS- on CS- 3000 .RTM. 3000 .RTM. 3000 .RTM.
Pre-Harvest Cell # 6.2 6.6 6.9 (.times.10.sup.8) Viability (%) 95
100 96 # cells/ml (.times.10.sup.6) 1.0 1.1 1.2 Post-Harvest Cell #
5 4.3 5.1 (.times.10.sup.8) Viability (%) 90 97 97 % Recovery 81 65
74 Lactate (g/L) 0.918 0.683 0.683 Glucose (g/L) 1.88 2.15 2.1 %
Cells in S + G2/M 57.8 55.5 62.5 CD Marker Profile % CD3+ 82 82 84
% CD3+ CD4+ 62 57 64 % CD3+ CD8+ 21 25 20 % CD3- CD16+ 2 5 2 % CD3+
CD56+ 1 2 3 % CD3- CD56+ 3 6 3 % CD3+ CD25+ 96 82 90 % CD3+ CD28+
93 76 80 % CD3+ HLA-DR+ 41 57 41 % CD3- HLA-DR+ 10 10 8
Retroviral Transduction of T-Cells
[0156] Moloney Murine Leukemia Virus (MMLV)-derived retroviral
supernatant (DAHSVTK9A) (3.9.times.10.sup.7 CFU/ml; Viagene Corp.,
San Diego, Calif.) was purchased from the manufacturer. The
supernatant preparation was added to the OKT-3-activated
mononuclear cells at a multiplicity of infection (MOI) of at least
about 3:1 to greater than about 5:1 according to the procedures set
forth below.
[0157] The retroviral supernatant preparation was stored at
-70.degree. C. Just prior to use, the supernatant preparation was
thawed aseptically in a 37.degree. C. water bath with gentle
agitation. The retroviral material was injected immediately into
the Lifecell.RTM. Flask, ensuring fluid-to-fluid contact. A new
syringe and needle were used for each Lifecell.RTM. Flask.
[0158] Supernatant preparations with a titer of less than
1.5.times.10.sup.7 cfu/ml were added directly to the Lymphocyte
Transduction Media. In cases where the titer of the supernatant was
less than about 1.5.times.10.sup.6, the retroviral material was
applied directly to the cells without further dilution with AIM V
medium. In this situation, the Protamine sulfate and rIL-2 were
added directly into the retroviral supernatant without the addition
of AIM V, L-Glutamine, or FBS. Supernatant preparations with a
titer of greater than about 1.5.times.10.sup.7 cfu/ml were
delivered to Lifecell.RTM. Flasks containing cells and Transduction
Media.
[0159] A sample of the supernatant preparation was reserved for
immediate titration using the murine cell line NIH3T3, the human
cell lines HT1080 and 143B, and the canine cell line Cf2Th. The
particular indicator cell line used to assay a supernatant
preparation depends upon the host range of the viral vector being
analyzed. Titers were determined using the general method of Cepko,
Current Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-Interscience, New York (1992), which is herein incorporated
by reference, except that the titer was determined by counting the
number of cells per plate on the first and second day of the assay
to more accurately determine the titer.
5TABLE 5 PARAMETERS FOR CELLS TO BE TRANSDUCED DONOR DONOR DONOR
Transduction A05 A06 A07 # Cells Transduced (.times.10.sup.8) 1.5
1.5 1.5 # Cells/ml set up (.times.10.sup.6) 0.5 0.5 0.5
Optional Post-Transduction Cell Expansion #1
[0160] Transduced mononuclear cells can be optionally expanded
following transduction. The purpose of such an expansion is to
ensure that a sufficient number of transduced cells are present in
the culture and thus survive G418 selection. The G418 selection
step described below was performed without expansion if the total
cell number in the initial activated, transduced culture was
greater than about 3.times.10.sup.8 cells. For cell numbers less
than about 3.times.10.sup.8, the following post-transduction
expansion procedures should be followed.
[0161] During expansion, the cell count, viability, and glucose and
lactate concentrations were measured about every two days. The
glucose concentration in the culture should be greater than about
100 mg/dL and the lactate concentration in the culture should be
less than about 1.0 mg/ml for the sample to be used in later
procedures.
[0162] White blood cell counts were determined using a Sysmex.RTM.
K-1000 cell counter (TOA Medical Electronics, Kobe, Japan)
following the manufacturer's instructions, paying particular
attention to the following modifications. When the cell count
became greater than 99.9.times.10.sup.6/ml, samples were diluted
1:10 with D-PBS.
[0163] Lymphocyte Culture Media was prepared by adding nutritional
and growth supplements to the AIM V Media to produce final
concentrations of 10% FBS, 2 mM Glutamine and 60 IU/ml rIL-2.
Glucose and lactate concentrations were determined as described
above, and the media was used the same day it was prepared.
[0164] For a cell density of at least 2.times.10.sup.6 cells/ml,
transduced mononuclear cells were expanded by diluting them to
about 5.times.10.sup.5 cells/ml in Lymphocyte Culture Media in
Lifecell.RTM. Flasks. Samples which had less than about
5.times.10.sup.8 total white blood cells were dispensed at about
5.times.10.sup.5 cells/ml into 1L Lifecell.RTM. Flasks. Samples
which had greater than about 5.times.10.sup.8 total white blood
cells were dispensed at about 5.times.10.sup.5 cells/ml into 3L
Lifecell.RTM. Flasks. Cultures were incubated at 37.degree. C./5%
CO.sub.2 as described above. The results of the first expansion of
A05, A06 and A07 donor cells are set forth in Table 6.
6TABLE 6 POST-TRANSDUCTION EXPANSION #1 Post-Transduction Expansion
1 DONOR A05 DONOR A06 DONOR A07 Cell # (.times.10.sup.8) 4.2 3.9
3.9 # cells/ml (.times.10.sup.6) 1.4 1.3 1.3 Viability (%) 90 94 93
Reseeded at 0.5 0.5 0.5 # cells/ml (.times.10.sup.6) Total Volume
(ml) 840 780 780
Optional Post-Transduction Cell Expansion #2
[0165] For total cell numbers at the end of the first
post-transduction expansion step equal to or greater than
3.times.10.sup.8 cells, the second post-transduction expansion step
was omitted. When cell density became greater than about
2.times.1.sup.06 cells/ml, transduced mononuclear cells were
expanded by diluting them to about 5.times.10.sup.5 white blood
cells/ml in Lymphocyte Culture Media in 1L Lifecell.RTM. Flasks.
For expansion cultures which had a total number of less than about
5.times.10.sup.8 white blood cells, cells were dispensed and
diluted at a concentration of about 5.times.10.sup.5 cells/ml into
1L Lifecell.RTM. Flasks. Expanded cultures having a total number of
greater than about 5.times.10.sup.8 white blood cells were
dispensed and diluted at a concentration of about 5.times.10.sup.5
cells/ml into 3L Lifecell.RTM. Flasks.
[0166] Expanded cultures were incubated at 37.degree. C. with 5%
CO.sub.2 as described above. Cell number, viability, and glucose
and lactate concentrations of activated, transduced mononuclear
cell cultures were determined as described above about every two
days after the first post-transduction expansion. The results
obtained in the second expansion are set forth in Table 7.
7TABLE 7 POST-TRANSDUCTION EXPANSION #2 Post-Transduction Expansion
2 DONOR A05 DONOR A06 DONOR A07 Cell # (.times.10.sup.9) 1.8 1.6
1.5 # cells/ml (.times.10.sup.6) 2.2 2.1 2 Viability (%) 92 94 88
Lactate (g/L) 0.911 0.993 0.826 Glucose (g/L) 1.86 1.72 1.89
Reseeded at 0.5 0.5 0.5 # cells/ml (.times.10.sup.6) Total Volume
(ml) 3600 3200 3000
Harvesting of Expanded Cells
[0167] Cells were harvested when the total number of expanded cells
became greater than about 3.times.10.sup.9 cells. The transduced
mononuclear cells from donor A05 were harvested using the single
chamber method of the CS-3000.RTM. Plus following the
manufacturer's instructions as discussed above. The transduced
cells from donors A06 and A07 were harvested using a Autopheresis C
with a 2 .mu.m filter at 1200 rpm. Characteristics of the cells
harvested prior to G418 selection are set forth below in Table
8.
8TABLE 8 CHARACTERISTICS OF HARVESTED CELLS EXPANDED FOR G418
SELECTION DONOR A05 DONOR A06 DONOR A07 Method of Harvest Single
Autopheresis Autopheresis chamber C C on CS- 3000 .RTM. Pre-Harvest
Cell 2.9 2.6 2.6 # (.times.10.sup.9) Viability (%) 96 96 98 #
cells/ml 0.8 0.8 0.9 Post-Harvest Cell 2.3 1.5 2 #
(.times.10.sup.9) Viability (%) 98 97 98 % Recovery 79 58 78
Lactate (g/L) 0.368 0.418 0.41 Glucose (g/L) 2.63 2.51 2.51 CD
Marker Profile % CD3+ 97 99 99 % CD3+ CD4+ 54 51 45 % CD3+ CD8+ 45
50 56 % CD3- CD16+ 0.0000 0.0000 0.0000 % CD3+ CD56+ 1 1 3 % CD3-
CD56+ 0.0000 0.0000 0.0000 % CD3+ CD25+ 36 93 78 % CD3+ CD28+ 98 89
78 % CD3+ HLA-DR+ 61 71 69 % CD3- HLA-DR+ 1 1 2
G418 Selection
[0168] Harvested transduced mononuclear cells were diluted and
dispensed at a concentration of about 5.times.10.sup.5 cells/ml in
G418 Selection Media using manual distribution methods. Harvested
populations having less than about 5.times.10.sup.8 total white
blood cells were diluted and dispensed at a concentration of about
5.times.10.sup.5 cells/ml into 1L Lifecell.RTM. Flasks. Harvested
populations having greater than about 5.times.10.sup.8 white blood
cells were diluted and dispensed at a concentration of about
5.times.10.sup.5 cells/ml into 3L Lifecell.RTM. Flasks.
[0169] G418 Selection Media (10% FBS, 2 mM Glutamine, 0.8 mg/ml
G418 and 60 IU/ml rIL-2 in AIM V media) was prepared using
heat-inactivated FBS, 100.times.L-Glutamine, and rIL-2. G418 was
added at a concentration of 0.8 mg/ml active drug. G418 is
commercially available as a powder and is very stable. However,
because the potency of G418 varies considerably from lot to lot,
and is generally low (about 450 .mu.g/mg of powder), care must be
taken to note the specific activity of each lot and to apply the
drug at appropriate concentrations. As for other media, glucose and
lactate concentrations were determined, and the selection media was
used the same day it was prepared.
[0170] A small sample (5 ml) of the G418 Selection Media was
reserved for quality control testing using the closed-system
transfer methods discussed above. The cultures were incubated at
37.degree. C. with 5% CO.sub.2. The results obtained from this
procedure are set forth in Table 9.
9TABLE 9 G418 SELECTION Initiate G418 Selection DONOR A05 DONOR A06
DONOR A07 # Cells set up in G418 1.8 1.4 1.9 Selection
(.times.10.sup.9) # cells/ml set 0.5 0.5 0.5 up (.times.10.sup.6)
Total Volume (ml) 3600 2800 3800 Viability (%) 98 97 98 Monitor
Cells (At 2 Days) Total Cell # (.times.10.sup.9) 2.0 1.7 3.8 #
cells/ml (.times.10.sup.6) 0.7 0.6 1 Viability 94 91 95
Optional Feeding with Fresh Selection Media
[0171] If desired, the mononuclear cells are fed with fresh G418
Selection Media, prepared as discussed above, after about two days.
Cell density is maintained at about 5.times.10.sup.5 cells/ml.
Samples of the cell cultures can be taken prior to harvesting for
quality control testing.
[0172] Cells are harvested using a Fenwal.RTM. Plasma Extractor
following the manufacturer's instructions by centrifuging 600 ml
Transfer Pack Containers. To ensure proper cell pellet formation,
the Transfer Pack Container is filled to 600 ml. The cells are
harvested by centrifugation at 1200 rpm (300 g) for 15 minutes. The
Transfer Pack Container is gently removed from the centrifuge
holder, and the pellet is checked for the formation of a pellet. If
no firm pellet has been formed, the Transfer Pack Container is
centrifuged for an additional 10 minutes. The Fenwal.RTM. Plasma
Extractor is used to remove the supernatant.
[0173] The transduced and G418-selected mononuclear cells are
dispensed at concentrations of about 5.times.10.sup.5 cells/ml into
fresh G418 Selection Media. For samples with less than about
5.times.10.sup.8 white blood cells, mononuclear cells are dispensed
at a concentration of about 5.times.10.sup.5 cells/ml into 1L
Lifecell.RTM. Flask for a total final volume of about 200 to 400
ml. Samples having greater than about 5.times.10.sup.8 mononuclear
cells are dispensed at a concentration of about 5.times.10.sup.5
cells/ml into 3L Lifecell.RTM. Flasks for a total final volume of
preferably around one, but no more than about 1.5 liters. Cultures
are incubated at 37.degree. C. with 5% CO.sub.2.
Optional Neo Selection Expansion #1
[0174] Cell count, viability, glucose and lactate concentrations
are determined on about the second day of G418 selection, using the
Sysmex.RTM. K-1000 and the YSI Model 2700 as discussed above. If
the cell density exceeds about 2.times.10.sup.6 cells/ml on the
second day following G418 selection, transduced mononuclear cells
are expanded by diluting them to about 5.times.10.sup.5 cells/ml in
G418 Selection Media, prepared as discussed above. If the cell
density is less than about 2.times.10.sup.6 cells/ml, culturing is
continued and the cell density monitored on the third day after
G418 selection.
[0175] Samples with less than about 5.times.10.sup.8 total white
blood cells are dispensed at about 5.times.10.sup.5 cells/ml into
1L Lifecell.RTM. Flasks. Samples with greater than about
5.times.10.sup.8 total white blood cells are dispensed at about
5.times.10.sup.5 cells/ml into 3L Lifecell.RTM. Flasks. Cultures
are incubated at 37.degree. C. with 5% CO.sub.2.
Harvesting
[0176] About four days following the initiation of G418 selection,
G418 selection media was removed from the cell culture by gently
harvesting the selected mononuclear cells using one of the
following Options 1, 2, or 3.
Harvesting by Centrifugation
[0177] G418 Selection Media was removed using the Fenwal.RTM.
Plasma Extractor following the manufacturer's instructions as
discussed above.
Harvesting by Ficoll.RTM. -Hypaque
[0178] For cultures having a viability of greater than 50%, G418
Selection Media and dead cells were removed with the CS-3000.RTM.
Plus using Ficoll-Hypaque.RTM. separation procedures according to
the manufacturer's instructions.
[0179] A 1000 ml Lifecell.RTM. Flask containing AIM V Media and
fitted with a Plasma Transfer Set with Spike and Needle Adapter was
connected to the Saline and Vent lines using the Sterile Tubing
Welder (one lead to the vent and another lead to the saline line).
A 600 ml Transfer Pack Container (for product) and a 2000 ml
Transfer Pack Container (for waste) were aseptically docked to the
Plasma Collect line using the "Y" tubing leads obtained from an 800
ml Transfer Pack Unit with two couplers. The roller clamps on the
Saline, Vent, and Plasma Collect lines were then opened. The Inlet,
Return, and ACD line roller clamps were closed.
[0180] Receipt of the apheresis product was performed using the
following procedure. When spiking Flasks using spike couplers, it
is important to ensure that spikes are securely inserted, as
improper insertion may result in the formation of air blocks. The
cells were gently resuspended using the sterile tubing welder and
transferred to a new 600 ml Transfer Pack Container holding 200 ml
of medium. An appropriate amount of saline/ACD from the original
Flask was reserved to determine cell count, viability, and
sterility. Cell counts were performed using the Sysmex.RTM. K-1000
as discussed above. Percent viability of the cells was also
determined. The Product/Saline Flask was connected to one lead of a
Three Lead-Type Blood Solution Recipient Set. The second lead was
attached to a 500 ml Flask of saline ACD. Lastly, the third lead
was spiked into one of the two female ports of a Sepacell.RTM. Lab
Adapter Set. The other female port a Plasma Transfer Set was fitted
with a needle adapter, and the needle adapter was inserted into the
Ficoll.RTM. Flask and the connection secured with adhesive tape.
The spiked end of the Sepacell Lab Adapter Set was then heat
sealed. A "Y" tubing lead obtained from an 800 ml Transfer Pack
Unit was welded to the long lead (retain roller clamp) of the 3
Lead Type Blood-Solution Recipient Set containing the Flasks. The
"Y" tubing lead was spliced into line 5 (component rich plasma
line) of the Apheresis Set using the Sterile Tubing Welder. The
Ficoll.RTM. separation procedure was performed essentially
according to the manufacturer's instructions.
Harvesting by Autopheresis C
[0181] G418 Selection Media was removed using the Autopheresis C
following the manufacturer's instructions as described above. The
results of harvesting the G418-harvested using the above-described
three options are set forth below in Table 10.
10TABLE 10 G418-SELECTED CELLS Method of Harvest Centrifugation
Autopheresis Autopheresis Extraction C C Pre-Harvest Cell 3.5 1.9
6.1 # (.times.10.sup.9) Viability (%) 88 74 95 # cells/ml
(.times.10.sup.6) 1 0.7 1.6 Post-Harvest Cell 3.2 2.9 7.5 #
(.times.10.sup.9) Viability (%) 90 88 96 % Recovery 91 153 123
Lactate (g/L) 0.363 0.362 0.66 Glucose (g/L) 2.45 2.43 2.1 CD
Marker Profile % CD3+ 99 100 100 % CD3+ CD4+ 45 43 44 % CD3+ CD8+
57 59 56 % CD3- CD16+ 0 0 0 % CD3+ CD56+ 1 1 9 % CD3- CD56+ 0 0 0 %
CD3+ CD25+ 16 62 54 % CD3+ CD28+ 95 82 75 % CD3+ HLA-DR+ 50 52 57 %
CD3- HLA-DR+ 1 0 1
[0182] Isolated, transduced, G418-selected mononuclear cells were
resuspended at a concentration of about 1.times.10.sup.6 cells/ml
in Lymphocyte Culture Media in new Lifecell.RTM. Flasks using
Flask-to-Flask transfer methods, reserving samples for quality
control testing. Another small sample (6 ml) was retained from the
culture media using the closed-system distribution methods for
Sysmex.RTM. K-1000 analysis, cell viability, sterility testing, and
Southern Analysis to determine transgene integration.
[0183] Cultures having less than about 5.times.10.sup.8 mononuclear
cells were dispensed at about 1.times.10.sup.6 cells/ml into 1L
Lifecell.RTM. Flasks and were incubated at 37.degree. C. with 5%
CO.sub.2.
Post-Selection Expansion
[0184] Post-selection expansion was performed as follows. When the
concentration of cells in the culture reached at least about
2.times.10.sup.6 cells/ml, the transduced mononuclear cells were
expanded by diluting them to a concentration of about
1.times.10.sup.6 cells/ml in Lymphocyte Culture Media, prepared as
described above, into Lifecell.RTM. Flasks. Cultures having less
than about 5.times.10.sup.8 mononuclear cells were dispensed at
about 1.times.10.sup.6 cells/ml into 1L Lifecell.RTM. Flasks while
cultures having greater than about 5.times.10.sup.8 mononuclear
cells were diluted and dispensed at about 1.times.10.sup.6 cells/ml
into 3L Lifecell.RTM. Flasks. For cell concentrations less than
about 2.times.10.sup.6 cells/ml, no expansion was performed. Cell
count, viability, and glucose and lactate concentrations were
determined at least every two days.
Cryopreservation of Cells and Thawing Cells
[0185] About three days after expanding the G418-selected white
blood cells, mononuclear cells were harvested using 1 of the 3
harvesting options as described above. Before the cells were
harvested, a 35 ml sample of culture media was removed for cell
count testing on Sysmex.RTM. K-1000; viability testing; analysis of
glucose and lactate concentrations with the YSI Model 2700
instrument; cryopreservation; and Southern Blot analysis. A portion
of the final harvested cell product and culture medium was retained
for quality control testing.
[0186] Harvested cells were cryopreserved at a concentration of up
to 50.times.10.sup.6 cells/ml in 10% DMSO with 20% human AB serum
or autologous plasma in Cryocyte.RTM. containers. The cells were
frozen in a Control Rate Freezer with a 4.degree. C. starting
temperature that dropped at a rate of -1.degree. C./minute to
-60.degree. C. and then at a rate of -10.degree. C./minute to
-90.degree. C.
[0187] Samples were stored in the vapor phase of liquid
N.sub.2.
Determination of HSV-TK Gene Copy Number in Transduced T-Cells
[0188] Quantitative Southern Blotting was used to determine the
gene copy number per cellular genome in the transduced clinical and
analytical scale cell populations using the procedure described
below. The variance between assay I and II reveals significant
variation in the gene copy number prior to and immediately
following G418 selection. For example, three days after G418 was
removed, gene copy number increased by two- to ten-fold, indicating
that the selection conditions enriched for HSV-TK transduced cells.
In assays I and II, transduction efficiencies for cells transduced
and grown on a clinical scale displayed a comparable gene copy
number of between 0.5 and 1 (see Table 11).
[0189] DNA was prepared and analyzed by Southern blotting as
follows. Briefly, genomic DNA mini-preps were performed using a
Qiagen Kit (Quiagen, Inc., Chatsworth, Calif.) according to the
manufacturer's instructions. Genomic DNA was solubilized in pH 8.0
at 65.degree. C. and quantified using a TKO100 device (Hoefer
Scientific Instruments, San Francisco, Calif.) according to the
manufacturer's instructions.
11TABLE 11 SUMMARY OF QUANTITATIVE SOUTHERN BLOT RESULTS FOR
CLINICAL SCALE TRANSDUCTION IDENTIFICATION COPY NUMBER Sample
Apheresis Plate/ Number Number Flask Code Day Assay I Assay II 1
A05 Flask PACT 4 0.08* 0.00 2 " " PTE 8 0.31 0.06 3 " " SE 12 ND
0.10 4 " " FP 15 0.59 0.48 5 A05 Plate PTE 4 0.48 0.19 6 " " FP 11
0.46 0.20 7 A06 Flask PACT 4 ND 0.00 8 " " PTE 8 0.09 0.13 9 " " SE
12 ND 0.10 10 " " FP 15 1.01 0.83 11 A06 Plate PTE 4 ND 0.10 12 " "
FP 11 0.70 0.13 13 A07 Flask PACT 4 0.00 0.00 14 " " PTE 8 ND 0.26
15 " " SE 12 0.59 0.24 16 " " FP 15 0.52 0.66 17 A07 Plate PTE 4
0.95 0.37 18 " " FP 11 1.02 0.47 *= No band was visible on the gel
ND = Not determined due to insufficient DNA yield or sample
degradation PACT = Post-OKT-3 Activation Step PTE = Post
Transduction Expansion Step SE = Selected Cells, day of G418
removal FP = Final Product, 3 days post-G418 removal
[0190] Copy number standards were prepared based on a standard of 5
mg of genomic peripheral blood lymphocyte (PBL) DNA per lane. These
standards were prepared fresh in siliconized microfuge tubes. The
standard stock solution contained 20 ng/ml of pLTIN/Nhe I (7068bp
plasmid/.about.4 kb fragment). One microliter of each copy number
dilution was added to 5 .mu.g of Nhe I-digested huPBL DNA.
Preparation of the copy number standards is summarized below in
Table 12.
12TABLE 12 DILUENT COPY SAMPLE (TE) CONCENTRATION NO. 1.18 ml of 20
ng/ml 48.82 .mu.l 471.2 pg/ml 80 2.5 ml of 471.2 pg/ml 47.5 .mu.l
23.56 pg/.mu.l 4 20 ml of 23.56 pg/.mu.l 20 ml 11.78 pg/ml 2 20 ml
of 11.78 pg/ml 20 ml 5.89 pg/ml 1 20 ml of 5.89 pg/ml 20 ml 2.95
pg/ml 0.5 20 ml of 2.95 pg/ml 20 ml 1.47 pg/ml 0.25 20 ml of 1.47
pg/ml 20 ml 0.736 pg/ml 0.125 20 ml of 0.736 pg/ml 20 ml 0.368
pg/ml 0.0625
[0191] Agarose electrophoresis in Tris-acetate buffer was used to
separate the components of these samples according to the general
method of Sambrook et al., Molecular Cloning: A Laboratory Manual,
2d ed., Cold Spring Harbor Laboratory Press (1989).
[0192] Southern blotting was performed on the electrophoresed
samples as described below. The DNA in the agarose gel was
de-purinated by incubating the gel in 0.25 N HCl for 20 minutes
under gentle agitation. The DNA in the agarose gel was denatured by
incubating in 0.5 N NaOH-1.5 M NaCl for 20 minutes under gentle
agitation. The gel was neutralized by shaking gently in 1 M
Tris-HCl pH 7.5-1.5 M NaCl for 20 minutes and the gel was
equilibrated by shaking gently in 20.times.SSC for 20 minutes.
[0193] DNA was transferred to a membrane by Southern blotting
essentially as described in Reed et al. (1985) Nuc. Acids Res.
13(20):7207-7221, which is incorporated herein by reference. The
moist membrane was crosslinked in a Stratalinker.RTM. (Stratagene,
La Jolla, Calif.) and air dried before hybridization.
[0194] The blot was pre-hybridized in 10 mg/ml denatured salmon
sperm DNA in QuikHyb (Stratagene, La Jolla, Calif.).
.sup.32P-labeled probes were prepared using a Prime-It kit
(Stratagene, La Jolla, Calif.) and hybridized in roller bottles or
in a hybridization oven. Membranes were washed in 2.times.SSC/0.1%
SDS at room temperature followed by washing five times with
0.1.times.SSC/0.1% SDS at 65.degree. C. for 30 minutes in a
hybridization oven. Autoradiography was performed with an
intensifying screen at -70.degree. C. The autoradiographic signals
were quantitated using densitometry.
EXAMPLE 2
Clinical Scale Retroviral Transduction
[0195] This example demonstrates that about 2.times.10.sup.9 to
2.times.10.sup.11 transduced cells can be produced in a closed
system. Based on animal tumor models with adoptively transferred
Lymphokine Activated Killer (LAK) cells, it is estimated that
approximately 1.times.10.sup.11 cells are required to treat human
tumors (Rosenberg et al. (1990) New England Journal of Medicine
323:570). The scale-up procedures described in the present Example
generate about 2.times.10.sup.11 retrovirally-transduced T-cells in
a closed system. As with Example 1, all Fenwal.RTM. products are
available from Fenwal Division, Baxter Healthcare, Deerfield,
Ill.
[0196] This procedure follows the procedure set forth in Example 1,
unless the instant Example calls for a different or modified
procedure.
Isolation of Mononuclear Cells by Ficoll-Hypaque.RTM. Procedure
[0197] At least 4.times.10.sup.9 white blood cells are obtained
from an apheresis product and subject to Ficoll-Hypaque.RTM.
purification as described in Example 1. Purified product thus
obtained is not used if it contains fewer than 2.times.10.sup.9
white blood cells.
OKT-3 Activation of T Lymphocytes
[0198] OKT-3 activation of T-cells is carried out substantially as
in Example 1. Due to the increased volume, three-liter
Lifecell.RTM. Flasks were substituted for one-liter Lifecell.RTM.
Flasks. In addition, cells and medium are transferred among
Lifecell.RTM. Flasks and the various instruments described below
using a Baxter-Fenwal.RTM. Solution Transfer Pump (Fenwal.RTM.
#6455) according to the manufacturer's directions. Lymphocyte
Activation Media is prepared as follows using a 10 L AIM V media
flask. A Lifecell.RTM. Transfer Set (Fenwal.RTM. #4C2474) is
installed on the Solution Transfer Pump according to the package
instructions, as is a Lifecell.RTM. Filter Adapter Set (Fenwal.RTM.
#4C2475). A Sepacell.RTM. Laboratory Adapter Set (Fenwal.RTM.
#4C2459) is inserted into the ten-liter media Flask (adapter sets
are then piggybacked, if necessary, to provide additional female
ports). A Plasma Transfer Set (Fenwal.RTM. #4C2243) is inserted
into one of the female ports of the Lifecell.RTM. Adapter set. The
other spike of the Plasma Transfer Set is inserted into the lead
tube of the Lifecell.RTM. Adapter Set, assuring that all clamps are
closed. A 2000 ml Transfer Pack Container (Fenwal.RTM. #4R2041) is
hung on the final container hook of the Solution Transfer Pump and
a final container connector is inserted into the Transfer Set
junction.
[0199] In order to remove excess media from the ten-liter Aim V
Flask, the Solution Transfer Pump is programmed following
manufacturer's instructions to withdraw a volume of media equal to
the volume of supplements to be added with the specific gravity set
to 1.00. At the completion of the pumping cycle, the tubing leads
from the Transfer Pack Container and media Flask are heat sealed.
Lymphocyte activation media is prepared as in Example 1,
substituting a 2000 ml Transfer Pack Container for the 300 ml
container.
[0200] Cells are dispersed at a final concentration of about
5.times.10.sup.5 cells/ml in Lymphocyte Activation Media according
to the following ranges. For samples having less than about
5.times.10.sup.9 white blood cells; about 5.times.10.sup.5 white
blood cells/ml are dispensed with Lymphoid Activation Media to a
total volume of about 1L to about 1.5L per 3L Lifecell.RTM. Flask.
For samples with greater than about 5.times.10.sup.9 cells, about
7.times.10.sup.5 white blood cells/ml are dispensed with about 3L
to about 1.5L total volume per 3L Lifecell.RTM. Flask. The
preferred total volume in a 3L Lifecell.RTM. Flask is about 1 L,
with a maximum volume of about 1.5L. Lifecell.RTM. Flasks are
incubated on wire racks at about 37.degree. C. at about 5% CO.sub.2
for about 3 days.
Retroviral Transduction of T Lymphocytes
[0201] Activated T-cells are harvested using the double-chamber
method of the Fenwal.RTM. CF3000 Plus (Baxter Fenwal.RTM. #4R4538).
The double-chamber method uses the following material in addition
to that used for the single-chamber method: 600 ml Transfer Pack
Unit with coupler (Fenwal.RTM. #4R2023); a Lifecell.RTM. Flask
(1000 ml capacity; Fenwal.RTM. #4R2110); and a Plasma Transfer Set
with two couplers (Fenwal.RTM. #4C2243). An A35 chamber, instead of
a small volume collection chamber, is inserted into the collection
holder of the CS-3000.RTM. Plus. The priming procedure is as
described above in Example 1.
[0202] The following steps are substituted in harvest procedure of
Example 1 after the first two steps have been carried out. Two
Plasma Transfer Sets are connected to a 600 ml Transfer Pack
Container with coupler (the 600 ml Transfer Pack Container will be
used as a "pooling pack"). The Transfer Pack Container is connected
to the lead tube of the manifold(s). The Sterile Tubing Welder is
used to attach a "Y" tubing lead obtained from an 800 ml Transfer
Pack Unit (Fenwal.RTM. #4R2055) to one of the Plasma Transfer Sets.
The "Y" tubing is spliced into line 5 (component rich plasma line)
of the open system Apheresis Set. The other Plasma Transfer Set is
attached to the inlet line. The roller clamps are then opened on
both Plasma Transfer Sets. The saline and inlet clamps also are
opened.
[0203] The inlet line to the Pooling Pack is primed by squeezing
the prime media to push approximately 50 ml media to the pooling
pack. The inlet clamp is then closed. A hemostat is placed above
the junction on the component-rich plasma line. A blood pump
(approx. 20 ml/min) is turned on to prime the line to the Pooling
Pack. The Pooling Pack is filled with an additional 50 ml of media.
The blood pump is turned off and the hemostat placed below the
junction. The saline clamp is closed, and the vent and plasma
return clamps are opened. The centrifuge is started, and when it
reaches full speed (approximately 1600 rpm), the vent and plasma
return clamps are closed.
[0204] The plasma collect clamp is then opened, ensuring that the
Waste Flask is not occluded. Also, it is important to ensure that
the roller clamps on the Plasma Transfer Sets leading to the
pooling pack are open. The plasma flow direction switch is set to
forward, and the plasma flow rate control is turned to 10 ml/min,
ensuring that there is flow to the Waste Flask. The inlet and
plasma return clamps are opened, and the blood flow rate control
set to 10 ml/min, ensuring that there is flow to the Waste Flask.
All roller clamps are opened to the culture Flasks and the pooling
pack is allowed to fill. It is important to note that the roller
clamp on the wash media is kept closed. Also, it may be necessary
to place the Pooling Pack at a level below the culture Flasks. The
flow rates of both pumps are increased to full speed (approx. 87
ml/min). When all culture Flasks have been emptied and the Pooling
Pack is almost empty, all pumps are stopped, all clamps and closed
and the centrifuge is stopped. The centrifuge compartment and the
hemostat lines leading to each chamber are then opened. The
resultant pellets are resuspended by gently massaging the appended
Lifecell.RTM. Flasks. The Lifecell.RTM. Flasks are re-inserted into
their respective chambers and the centrifuge door is closed. The
roller clamp between the manifold set and the pooling pack is
closed. The media is washed and allowed to fill the Lifecell.RTM.
Flasks by opening the roller clamps, and the flasks rinsed by
inversion. The roller clamp is opened to the pooling pack.
[0205] The above procedure is repeated to restart the centrifuge
and the cells are washed by continuing to pump media. Once the
media is emptied from the Pooling Pack, all of the pumps, the
clamps and the centrifuge are turned off. The Lifecell.RTM. Flasks
in both chambers are sealed off and enough of a lead on each Flask
is retained to make a sterile connection. The Lifecell.RTM. Flasks
in the CS-3000.RTM. are sterilely connected in order to pool both
products into a Collection Flask. The Collection Flask is weighed
to determine volume using an empty Flask to zero the scale.
[0206] The remainder of the procedure is substantially similar to
that of Example 1, except that the three-liter Lifecell.RTM. Flasks
are substituted for one-liter Lifecell.RTM. Flasks. In addition,
the Lymphocyte Transduction Media was prepared from a ten-liter AIM
V media Flask using a Fenwal.RTM. Solution Transfer Pump. The steps
for the initial parameters for this Solution Transfer Pump are
discussed above for the Lymphocyte Activation Media. The
Lifecell.RTM. Flasks are then incubated overnight (rather than at
least twelve hours).
Cell Maintenance and Expansion
[0207] About twenty-four hours after the addition of the retrovirus
supernatant, the transduced white blood cells are harvested using
the double-chamber method of the CS-3000.RTM. Plus. The rest of the
procedure is substantially the same as Example 1, except that
three-liter Lifecell.RTM. Flasks are substituted for the one-liter
variety. For samples which have less than about 20.times.10.sup.9
white blood cells, about 5.times.10.sup.5 white blood cells/ml with
about 1L to about 1.5L of Lymphocyte Culture Media are dispensed to
each 3L Lifecell.RTM. Flask. For samples with greater than about
20.times.10.sup.9 white blood cells, about 7.times.10.sup.5 white
blood cells/ml with about 1L to about 1.5L of Lymphocyte Culture
Media are dispensed to each 3L Lifecell.RTM. Flask. The preferred
media volume for each three-liter Lifecell.RTM. Flask is about 1L,
with the maximum media volume being about 1.5L.
[0208] Lymphocyte Culture Media is prepared using a Fenwal.RTM.
Solution Transfer Pump, in a manner similar to that used to prepare
the Lymphocyte Activation Media and Retroviral
Supernatant/Transduction Media (e.g., a ten-liter AIM V media Flask
is manipulated using, in addition to the regular procedure, a
Lifecell.RTM. Transfer Set, a Lifecell.RTM. Filter Adapater Set and
a 2000 ml Transfer Pack Container).
[0209] The cell growth is aseptically monitored as described in
Example 1. When cell densities reach approximately 2.times.10.sup.6
cells/ml, the cells are split and reseed at about 5.times.10.sup.5
cells/ml in additional Lymphocyte Culture Media. The white blood
cells should be greater than about 90% viable, and the white blood
cell concentration should be greater than about 5.times.10.sup.5
cells/ml. The glucose concentration in the media should be greater
than about 100 mg/dL, and the lactate concentration should be less
than about 1.0 mg/ml. If the cells and media do not meet these
criteria, then they are not suitable for later procedures.
G418 Selection of Neo-Transduced T Lymphocytes
[0210] After the cells are isolated using the double-chamber method
on the CS-3000.RTM. Plus as described above, transduced white blood
cells were resuspended in G418 selection media (made using the
Fenwal.RTM. Solution Transfer Pump and a ten-liter Flask of AIM V).
The selection media and cells are transferred into three-liter
Lifecell.RTM. Flask using the Solution Transfer Pump according to
the manufacturer's instructions. For samples with less than about
20.times.10.sup.9 white blood cells, about 5.times.10.sup.5 white
blood cells/ml are dispensed with about 1L of G418 selection media
to yield about 1.5L total media volume per Lifecell.RTM. Flask. For
samples of transduced cells with greater than about
20.times.10.sup.9 white blood cells in each Lifecell.RTM. Flask,
about 7.times.10.sup.5 white blood cells/ml are dispensed and
enough G418 Selection Media added to yield a final volume of about
1L to about 1.5L total volume per Lifecell.RTM. Flask. The
preferred media volume is about 1L per 3L Lifecell.RTM. Flask with
the maximum volume of media being up to about 1.5L.
[0211] Cells can be fed with fresh G418 Selection Media after about
three days. Transduced white blood cells can be harvested using the
double chamber method on the CS-3000.RTM. as set forth above, or by
other acceptable methods. With some donor cells, it may be
necessary to split, dilute and reseed at the cells at about
5.times.10.sup.5 white blood cells/ml if the cell density exceeds
about 2.times.10.sup.6 white blood cells/ml during G418
selection.
[0212] About five days after initiation of the selection, selected
cells are harvested by washing gently in fresh Lymphocyte Culture
Media. Transduced white blood cells can be harvested using the
double chamber method using the CS-3000.RTM. Plus as discussed
above or by other acceptable methods.
[0213] The transduced, G418-selected white blood cells are
resuspended at about 1.times.10.sup.6 cells/ml in Lymphocyte
Culture Media. The G418-selected white blood cells are monitored
and cultured as above with the following variation. The viable cell
density is maintained at about 1.times.10.sup.6 white blood
cells/ml with frequent media exchanges until the resistant cells
have undergone several rounds of cell division, at which time
seeding density may be reduced to about 5.times.10.sup.5 white
blood cells/ml.
Cryopreservation and Subsequent Thawing of Cells
[0214] This procedure is carried out substantially as in Example 1,
except the Fenwal.RTM. Cell Harvester (Baxter Fenwal.RTM. #4R4960)
is used to harvest the transduced, G418-selected cells. The
Fenwal.RTM. Cell Harvester is used in conjunction with a
Fenwal.RTM. Mobile Work Station (Baxter Fenwal.RTM. #4R4962).
Components that were used with the Fenwal.RTM. Plasma Extractor in
Example 1 are also applicable to this Example. The Fenwal.RTM. Cell
Harvester is used as set forth in the operator's manual.
Cell Monitoring and Sampling Procedures
[0215] Generally, at a minimum, culture and cell monitoring is
performed on the initial product, and at the initiation and
completion of each processing step. The choice of assays will
depend on the equipment and resources available. Specific methods
of monitoring are set forth in Example 1.
EXAMPLE 3
Preparation of Retroviral Vector Backbones
[0216] This example describes the construction of several
retroviral backbones useful in the preparation of the gene transfer
vectors of the present invention.
[0217] A. Preparation of DKT-1 and pKT-3B vectors.
[0218] The Moloney murine leukemia virus (MoMLV) 5' long terminal
repeat (LTR) EcoRI-EcoRI fragment (including gag sequences) from
the N2 vector (Armentano et al. (1987) J. Virol. 61:1647-1650,
Eglitas et al. (1985) Science 230:1395-1398) is ligated into the
plasmid SK+(Stratagene, La Jolla, Calif.). The resulting construct
is designated N2R5. The N2R5 construct is mutated by site-directed
in vitro mutagenesis to change the ATG start codon to ATT
preventing gag expression. This mutagenized fragment is 200 base
pairs (bp) in length and flanked by PstI restriction sites. The
PstI-PstI mutated fragment is purified from the SK+plasmid and
inserted into the PstI site of N2 MoMLV 5' LTR in plasmid pUC31 to
replace the non-mutated 200 bp fragment. The plasmid pUC31 is
derived from pUC19 (Stratagene, La Jolla, Calif.) in which
additional restriction sites XhoI, BglII, BssHII and NcoI are
inserted between the EcoRI and SacI sites of the polylinker. This
construct is designated pUC31/N2R5gM.
[0219] A 1.0 kilobase (Kb) MoMLV 3' LTR EcoRI-EcoRI fragment from
N2 is cloned into plasmid SK+ resulting in a construct designated
N2R3-. A 1.0 Kb ClaI-HindIII fragment is purified from this
construct.
[0220] The ClaI-ClaI dominant selectable marker gene fragment from
the PAFVXM retroviral vector (Kriegler et al. (1984) Cell 38:483,
St. Louis et al. (1988) Proc. Natl. Acad. Sci. USA 85:3150-3154),
comprising a SV40 early promoter driving expression of the neomycin
(neo) phosphotransferase gene, is cloned into the SK+ plasmid. This
construct is designated SK+SV2-neo. A 1.3 Kb ClaI-BstBI gene
fragment is purified from the SK+SV2-neo plasmid.
[0221] KT-3B or KT-1 vectors are constructed by a three part
ligation in which the XhoI-ClaI fragment containing a gene of
interest, and the 1.0 Kb MoMLV 3' LTR ClaI-HindIII fragment are
inserted into the XhoI-HindIII site of pUC31/N2R5gM plasmid. This
gives a vector designated as having the KT-1 backbone. The 1.3 Kb
ClaI-BstBI neo gene fragment from the pAFVXM retroviral vector is
then inserted into the ClaI site of this plasmid in the sense
orientation to yield a vector designated as having the KT-3B
backbone.
[0222] B. Preparation of pBA-5a, pBA-5b, pBA-5c, pBA-9b and
pBA-8bL1.
[0223] Several modifications can be made to the retroviral vector
pKT-l which result in decreased sequence homology to the retroviral
gag/pol and envelope expression constructs. In addition, two stop
codons were introduced in the DNA sequence of the packaging signal
sequence in order to increase the safety of these vectors. The
resulting retroviral backbones are called pBA-5a, pBA-5b, and
pBA-5c. Further details on the construction of pBA-5a, pBA-5b and
pBA-5c can be found in co-owned U.S. patent application Ser. No.
08/721,327 and co-owned U.S. patent application entitled "Crossless
Retroviral Vectors," filed May 5, 1997 (attorney docket 1147.004)
both of which are hereby incorporated by reference.
Substitution of Nonsense Codons in the Extended Packaging Sequence
(.PSI.+)
[0224] Modification of the extended packaging signal (.PSI.+) was
conducted using PCR on the template KT-1 with primers that
introduce two stop codons in the extended packaging signal
sequence. In particular, the template pKT-1 contains the
modification ATT at the normal ATG start site of gag. Here the
start site was further modified to the stop codon, TAA, and an
additional stop codon TGA was added to replace the codon TTA at
position 645-647 of the sequence depicted in SEQUENCE ID NO:15 of
co-owned U.S. provisional application entitled "Methods for
Administration of Recombinant Gene Delivery Vehicles for Treatment
of Hemophilia and Other Disorders," filed Jun. 4, 1997 (attorney
docket 1155.004), which application is incorporated herein by
reference.
[0225] Briefly, two sets of PCR reactions were carried out on
pKT-1, each introducing one stop codon. The primers for the PCR
were designed such that the two PCR products had overlapping
regions and a splice-overlap extension PCR (SOE-PCR) was carried
out with the two PCR products in order to combine the two
introduced stop codons on one strand. The first set of
oligonucleotides introducing the change from ATT to TAA were:
5'-GGG AGT GGT AAC AGT CTG GCC TTA ATT CTC AG;
[0226] and
5'-CGG TCG ACC TCG AGA ATT AAT TC,
[0227] and the second set of oligonucleotides introducing the
change from TTA to TGA were:
5'-CTG GGA GAC GTC CCA GGG ACT TC;
[0228] and
5'-GGC CAG ACT GTT ACC ACT CCC TGA AGT TTG AC.
[0229] The flanking primers of the final 708 base pair PCR product
introduced AatII and the XhoI sites, at the 5' and 3' ends,
respectively.
[0230] The ends of the 708 base pair product were blunted and
phosphorylated, and the product introduced into the SmaI and EcoRV
digested vector pBluescript SK-(Stratagene, San Diego, Calif.). The
resulting plasmid was designated pBA-2.
Removal of Retroviral Sequences Upstream and Downstream from the 3'
LTR and Upstream and within the 5' LTR
[0231] Retroviral envelope sequence was removed upstream of the 3'
LTR between the ClaI site and the TAG stop codon of the envelope
coding sequence. The DNA sequence was modified by PCR such that the
TAG stop codon was replaced by a ClaI site and the 97 nucleotides
upstream from this new ClaI site to the original ClaI site were
deleted, as well as the 212 base pairs of retroviral sequence
downstream of the 3' LTR.
[0232] Briefly, the following two oligonucleotides were used for
the PCR:
5'-CAT CGA TAA AAT AAA AGA TTT TAT TTA GTC;
[0233] and
5'-CAA ATG AAA GAC CCC CGC TGA C,
[0234] and the template was pKT-1. The PCR product was cloned into
PPCRII (Invitrogen, San Diego, Calif.) using the TA cloning kit
(Invitrogen, San Diego, Calif.) and called pBA-1.
[0235] Subsequently, pBA-2 was digested with XbaI and AatII which
deleted a part of the multiple cloning site and the 780 base pair
fragment from NheI to AatII from pKT1 was cloned into the
linearized vector, resulting in the plasmid pBA-3. Plasmid pBA-3
combined the shortened 5' LTR with the above-described packaging
region including the two introduced stop codons.
[0236] The pBA-1 construct was then digested with ClaI and ApaI to
obtain a 640 base pair fragment that was cloned into the ClaI and
ApaI-digested pBA-3, resulting in the plasmid pBA-4. This plasmid
combines the above- described 5' LTR and the packaging signal with
the 3' LTR.
[0237] pBA-4 was digested with ApaI and EcoRI, blunt-end modified,
and religated in order to remove extraneous 3' polylinker sites,
resulting in plasmid pBA-5a.
[0238] Subsequently, pBA-5a was cut with NotI (blunted) and EcoRI,
and introduced into Sm&I and EcoRI-digested pUC18 (GIBCO/BRL,
Gaithersburg, MD) resulting in pBA-5b. This construct moved the
retroviral vector from a pBluescript backbone into an alternate
pUC18 vector.
[0239] pBA-5c is constructed in identical manner to pBA-5b, except
that the XhoI/ClaI multicloning site was introduced into
pUC-19.
[0240] Several further modifications to the retroviral vector
pBA-5b were carried out to provide a vector with multiple unique
restriction enzyme sites for convenient cloning. In order to
prepare the pBA-9b vector, the herpes simplex virus thymidine
kinase (HSVTK) gene was retrieved by digesting the pBH-1 construct
with XhoI and EcoRI, resulting in a 1.2 Kb fragment. (pBH-1 was
prepared as described in International Publication No. WO 91/02805,
entitled "Recombinant Retroviruses Delivering Vector Constructs to
Target Cells," which is hereby incorporated by reference.) The
neomycin gene driven by the SV40 promoter was retrieved by
digesting pKT-1 with EcoRI and BstBI, resulting in a 1.3 Kb
fragment. Both fragments were cloned into a XhoI and Cla-digested
pBA-5b, resulting in the retroviral vector pMO-TK.
[0241] The TK gene from the retroviral vector pMO-TK was isolated
as a XhoI-ClaI fragment and inserted into the XhoI and
ClaI-digested pBA-5b, resulting in the plasmid pBA-5bTK. In order
to delete the HindIII, SphI, PstI, SalI and HincII restriction
enzyme sites upstream of the 5' LTR, pBA-5bTK was digested with
HindIII and HincII, and the overhanging ends were removed using T4
polymerase and the blunt ends ligated using T4 DNA ligase. This
resulted in plasmid pTJBA-5bTK with 16 bases (TGC ATG CCT GCA GGT
C) removed from the region upstream of the 5' LTR. The plasmid
pTJBA-5bTK has a BamHI upstream of the 5' LTR. It is desirable to
remove this BamHI site since it is a common site used for cloning.
In order to destroy the BamHI site upstream of the 5' LTR, the
BamHI-containing TK gene in pTJBA-5bTK was replaced by the IL-2
gene via a XhoI and ClaI digest, resulting in plasmid pTJBA-5bIL-2.
The plasmid pTJBA-5bIL-2 was digested with BamHI, the ends filled
in with the Klenow fragment and religated, resulting in
pTJBA-5bIL-2 (BamHI del.).
[0242] In order to produce the plasmid pBA-9b, the IL-2 gene from
pTJBA-5bIL-2 (BamHI del.) is deleted via XhoI-ClaI digest, and
replaced with a polylinker that introduces a multiple cloning site
(MCS) and codes for the restriction enzyme sites 5'-XhoI ApaI BglII
NotI NruI SalI HindIII BamHI ClaI-3'. The sequences of the two
primers used to produce the linker are as follows:
5'-TCG AGG GGC CCA GAT CTG CGG CCG CTC GCG AGT CGA CAA GCT TGG ATC
CAT-3'
[0243] (as the primer for the positive strand); and
5'-CGA TGG ATC CAA GCT TGT CGA CTC GCG AGC GGC CGC AGA TCT GGG CCC
C-3'
[0244] (as the primer for the negative strand).
[0245] This example also describes several modifications of the
retroviral vector pBA-Sb which result in a vector coding for the
human placental alkaline phosphatase gene (PLAP), driven by the
SV40 promoter.
[0246] The plasmid pBA-8bL1 was constructed in a three-way ligation
using the following three fragments: (1) the NdeI-ClaI fragment
from pBA-5b (described above) containing the 3' LTR and the pUC18
backbone; (2) the ClaI-HindIII fragment from pCI-PLAP coding for
PLAP; and (3) the HindIII-NdeI fragment from pBA-6bL1 containing
the 5' LTR and the SV40 promoter. Plasmid pBA-6bL1 is based on
pBA-6b (described above) wherein the HIV env/rev coding region was
deleted via a XhoI-ClaI digest, and replaced with the L1 linker
coding for several restriction enzyme sites including XhoI at the
5' end and ClaI at the 3' end.
EXAMPLE 4
Preparation of DBA-5a, pBA-5b, and pBA-5c Retroviral Vectors
Expressing B Domain-Deleted Factor VIII
[0247] A B domain-deleted factor VIII cDNA fragment was obtained by
a XhoI/NotI digestion as described below. A retroviral vector
(pMBF8) expressing a B domain-deleted factor VIII is constructed
from the expression plasmid pSVF8-200 which is prepared as
previously described (Truett (1985) DNA 4:333 and U.S. Pat. No.
5,045,455). The pSVF8-200 plasmid was deposited with the ATCC on
Jul. 17, 1985, and assigned ATCC Accession No. 40190.
[0248] A DNA fragment encoding the B domain-deleted Factor VIII
molecule was obtained from pSVF8-302, which has a nine base pair
deletion in the 5' non-coding region after the poly G tail. Plasmid
SVF8-302 was constructed in a similar manner as pSVF8-200, which is
described in detail in Truett, supra, and in U.S. Pat. No.
5,045,455. Construction of pSVF8-302 is also described in U.S. Pat.
No. 5,595,886.
[0249] The procedure outlined below describes the construction of
retroviral vectors expressing a B domain-deleted Factor VIII
protein obtained from pSVF8-302. However, the same procedure can
also be used to construct such retroviral vectors from
pSVF8-200.
[0250] The full-length cDNA sequence of human factor VIII, and the
full-length amino acid sequence thereof are disclosed in co-owned
U.S. provisional application entitled "Methods for Administration
of Recombinant Gene Delivery Vehicles for Treatment of Hemophilia
and Other Disorders," filed Jun. 4, 1997 (attorney docket
1155.004). The cDNA sequence of the B domain-deleted SQN deletion,
and the SQN deletion amino acid sequence are also disclosed in the
above-reference provisional patent application.
[0251] Fragment 1, encompassing nucleotides 5500-6248 of pSVF8-200
(see FIG. 8 of Truett, supra), was obtained by VENT-PCR using
factor VIII primers encoding a PFlM1 site at the 3' end and the 5'
SQN sequence plus a HindIII site at the 5' end. The 5' primer
encompasses the region 2446-2460 of the 5' SQN and the 5144-5167
region just downstream of the 3' SQN sequence. Thus, this fragment
spans the sequence between the two SQN sites within the B domain
(positions 2461 and 5142). The particular primer sequences used
were:
5'-GAA GCT TCT CCC AGA ACC CAC CAG TCT TGA AAC GCC ATC;
[0252] and
5'-GTA CCA GCT TTT GGT CTC ATC AAA G.
[0253] Fragment 1 was blunt-end cloned into vector SK-that had been
cut with SmaI and dephosphorylated, forming pSK-Pfl. Fragment 2,
encompassing nucleotides 1190-2448, was isolated following HindIII
digestion and cloned into the HindIII site of SK-Pfl to form
SK-Pfl-Hind. The orientation of the insert was determined using
AccI and PstI digests. pSVF8-200 was digested with HpaI and
religated to remove two small HpaI fragments 3' to the factor VIII
cDNA insert, forming pF8-300-del-Hpa. The remaining HpaI site was
converted to a NotI site using NotI phosphorylated linkers, forming
F8-300-Hpa/Not. Fragment 3, encompassing nucleotides 5885-7604, was
isolated after a PflMI and NotI digestion, and cloned into
SK-Pfl-Hind following PflMI and NotI digestion of the latter to
form pF8:213. Fragment 4 (encompassing nucleotides 104-133 to 1204)
was obtained following VENT-PCR of PSV7dF8-300 with primers
containing 5' XhoI and 3' AccI sites respectively. The 5' primer
encompasses nucleotides 104-133, and the 3' primer encompasses
nucleotides 1200-1224. pF8:213 was digested sequentially with XhoI
followed by AccI, and ligated to Fragment 4 which was digested with
XhoI and AccI, to provide pF8:4213. The primer sequences for the 5'
UT and XhoI primers were:
5'-CTC CTC GAG CTA AAG ATA TTT TAG AGA AGA ATT AAC;
[0254] and
5'-TTC CTC TGG ACA GCT GTC TAC TTT G.
[0255] The above-described modified cDNA is cloned into the pMBA
backbone (also described above) which has been digested with XhoI
and NotI. Similarly, the cross-less backbones pBA-9b, pBA-5a,
pBA-5b and pBA-5c are modified by linearizing with ClaI, blunt-end
modifying, and religating in the presence of NotI phosphorylated
linkers. The modified cDNA fragment is cloned into the XhoI/NotI
linearized vectors.
EXAMPLE 5
Construction of Recombinant Adeno-Associated Virus (rAAV) Vectors
that Express the Heavy and Light Chains of Human Factor VIII
[0256] This example describes the construction of two rAAV gene
transfer vectors, one expressing the light chain, and the other the
heavy chain of human factor VIII. Both chains contain the Factor
VIII leader sequence and a variable amount of the B domain.
[0257] To clone the heavy chain, a region of the Factor VIII gene,
from 174 bp 5' of the ATG, to amino acid 745, was amplified by PCR.
This fragment includes the entire heavy chain and the first five
amino acids of the B domain. The oligos used were:
(forward) 5'-CAC CGT CGT CGA CTT ATG CT-3';
[0258] and
(reverse) 5'-GAC CGT CGA CTC AAT TCT GGG AGA AGC TTC TTG G-3'.
[0259] The plasmid used as a template in the PCR reaction was
pCMVKmHSTB, a B domain-deleted factor VIII expression construct.
The amplified fragment was digested with SalI, and cloned into a
CMV expression vector, pCMVKmLINK digested with SalI and XhoI. This
plasmid was called pCMVKm90H. pCMVkMLINK is an expression vector
containing the CMV promoter/intron, a polylinker for cloning genes
of interest, and a bovine growth hormone polyadenylation signal. To
make a rAAV vector expressing the heavy chain, pCMVKm90H was
digested with SalI and BamHI, the BamHI site was filled in with T4
DNA polymerase, and this fragment was cloned into the rAAV vector
pKm201CMV-CI digested with SalI and EcoRV. pKm201CMV-CI contains
the inverted terminal repeats of AAV, the CMV promoter, the
chimeric intron from pCI (Promega, Madison, Wis.), and the bovine
growth hormone polyadenylation signal. The final AAV vector was
called pKm201-90H.
[0260] To clone the light chain, the factor VIII 5' untranslated
and leader region sequences were amplified using the following
primers:
(forward) 5'-CAC CGT CGT CGA CTT ATG CT-3';
[0261] and
(reverse) 5'-CAA CGC TCG AGA AGC AGA ATC GCA AAA GGC-3'.
[0262] Again, pCMVKmHSTB was used as a template in the PCR
reaction. The amplified region includes sequences from 174 bp
upstream of the ATG, to amino acid 19 of factor VIII. This fragment
was digested with XhoI and SalI and cloned into pCMVKmLINK digested
with XhoI and SalI. This plasmid was called pCMVKmF8L (for factor
VIII leader). To amplify the light chain, the following primers
were used:
(forward) 5'-TCG GCT CGA GGC ATC AAC GGG AAA TAA CTC GT-3';
[0263] and
(reverse) 5'-CCG ACT CGA GTC AGT AGA GGT CCT GTG CCT C-3'.
[0264] Again, pCMVkMHSTB served as the template for the PCR. The
amplified fragment included sequences from amino acid 1645 of
factor VIII to the STOP codon after amino acid 2332. This included
the last four amino acids of the B domain and the complete light
chain. This fragment was digested with XhoI and cloned into the
XhoI site of pCMVKmF8L. This resulted in a light chain construct
containing the factor VIII leader which was called pCMVKm80L.
pCMVKm80L was digested with SalI and BamHI to remove the light
chain construct, and this fragment was cloned into pKm201CMV-CI
digested with SalI and BamHI to generate pKm201-80L.
EXAMPLE 6
Co-Infection of Cells with rAAV Vectors Expressing the Heavy and
Light Chains of Factor VIII Results in the Production of
Biologically Active Factor VIII
[0265] The heavy and light chain constructs, pKm201-80L and
pKm201-90H, were packaged following standard procedures for the
production of rAAV (Zhou et al. (1994) J. Exp. Med. 179:1867-1875).
rAAV was purified as described (Wang et al. (1995) Proc. Natl.
Acad. Sci. USA 92:12416-12420) and used to infect 293 cells plated
in 6-well plates. Supernatants of infected cells were collected 48
h after infection and assayed for biologically active factor VIII
by Coamatic Factor VIII (KabiVitrum, Stockholm), following the
manufacturer's instructions. Normal human plasma (George King
Bio-Medical, Inc., Overland Park, Kans.) was used to generate a
standard curve. Cells were infected at a multiplicity of infection
(MOI) of 6000 rAAV particles per cell. The experiment was done both
with and without the addition of etoposide (0.3 M) to the medium.
Etopiside has been shown to increase the transduction efficiency of
rAAV vectors (Russell et al. (1995) Proc. Natl. Acad. Sci. USA
92:51719-51723). As a result of the study, co-infection of rAAV-80L
and rAAV-90H resulted in the production of biologically active
factor VIII. The amount of factor VIII was increased in the
presence of etoposide.
EXAMPLE 7
Construction of a Retroviral Vector Expressing Human LDL
Receptor
[0266] This experiment describes the production of a retroviral
gene transfer vector expressing human LDL-receptor. The human
LDL-receptor expression plasmid pLDLR17 was obtained from Bev
Davidson, at the University of Iowa. Alternatively, the expression
plasmid can be prepared as described by (1989) J. Biol. Chem.
264:21682-88. The 5' fragment was reconstructed using VENT-PCR. The
3' primer contained a XhoI site, and the 3' primer encompasses the
unique EcoRI site within the LDLR cDNA. The EcoRI-digested 5'
fragment was subcloned into Bluescript SK- and cut with SmaI and
EcoRI. The SmaI site at the 3' end of LDLR cDNA in LDLR17 was
modified using a NotI linker to yield pLDLR17-S/N. The two
fragments (the 5' fragment: XhoI to EcoRI, and the 3' fragment:
EcoRI to NotI) were ligated to pBA-6b which was digested with XhoI
and NotI. The sequences of the PCR primers were:
5'-GCG ACT CGA GCA TGG GGC CCT GGG GC;
[0267] and
5'-GCA CTG GAA TTC GTC AGG GCG.
[0268] The resulting vector was named p6b-LDLR.
[0269] A high titer DA producer clone for p6b-LDLR was selected
under G418. The G418 vector titer in the supernatant was around
2.times.10.sup.7 cfu/ml. Expression in target cells in vitro was
demonstrated to be comparable to normal levels using either a
Western blot or a functional assay.
EXAMPLE 8
Human Alpha 1 Antitrypsin Retroviral Vectors for the Treatment of
Antitrypsin Deficiency
[0270] This example describes the preparation of a retroviral gene
transfer vector encoding human .alpha..sub.1-antitrypsin. The human
.alpha..sub.1-antitrypsin cDNA clone was obtained from the ATCC
(Clone #256976). The plasmid was digested with EcoRI and blunted
using T4 DNA polymerase large fragment (Klenow). The fragment
containing the cDNA is cloned into the SrfI linearized pBA-9 vector
to produce the provector pBA9-AAT. An oxidation resistant cDNA
clone prepared as described in U.S. Pat. 4,732,973 was digested
with restriction enzymes and ligated to pBA-5b.
[0271] Accordingly, methods for genetically modifying a population
of T cells, and gene transfer vectors for carrying out the
modifications are disclosed. Although preferred embodiments of the
subject invention have been described in some detail, it is
understood that obvious variations can be made without departing
from the spirit and the scope of the invention as defined by the
appended claims.
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