U.S. patent application number 09/899479 was filed with the patent office on 2002-08-08 for genetic modification of primate hemopoietic repopulating stem cells.
Invention is credited to Einerhand, Markus Peter Wilhelmus, Valerio, Domenico.
Application Number | 20020106795 09/899479 |
Document ID | / |
Family ID | 8224665 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106795 |
Kind Code |
A1 |
Einerhand, Markus Peter Wilhelmus ;
et al. |
August 8, 2002 |
Genetic modification of primate hemopoietic repopulating stem
cells
Abstract
Genetic modification of pluripotent hemopoietic stem cells of
primates (P-PHSC) by transduction of P-PHSC with a recombinant
adeno-associated virus (AAV). The genome of the recombinant AAV
comprises a DNA sequence flanked by the inverted terminal repeats
(ITR) of AAV. The DNA sequence will normally comprise regulatory
sequences that are functional in hemopoietic cells and, controlled
by these regulatory sequences, a sequence coding for a protein or
RNA with a therapeutic property when introduced into hemopoietic
cells. Preferred examples of DNA sequences are the human lysosomal
glucocerebrosidase gene, a globin gene from the human .beta.-globin
gene cluster, a DNA sequence encoding an RNA or protein with
anti-viral activity, the .alpha.1-antitrypsin gene and the human
multidrug resistance gene I (MDRI). The invention provides for
effective gene therapy with PHSC of primates, particularly
humans.
Inventors: |
Einerhand, Markus Peter
Wilhelmus; (Amsterdam, NL) ; Valerio, Domenico;
(Leiden, NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
8224665 |
Appl. No.: |
09/899479 |
Filed: |
July 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09899479 |
Jul 5, 2001 |
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09326032 |
Jun 4, 1999 |
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09899479 |
Jul 5, 2001 |
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PCT/NL97/00631 |
Nov 19, 1997 |
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Current U.S.
Class: |
435/372 ;
435/456 |
Current CPC
Class: |
C12N 15/86 20130101;
A61K 48/00 20130101; C07K 14/805 20130101; C12N 2750/14143
20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/372 ;
435/456 |
International
Class: |
C12N 005/08; C12N
015/861 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 5, 1996 |
EP |
96203444.3 |
Claims
We claim:
1. A primate pluripotent hemopoietic stem cell (P-PHSC), said
P-PHSC produced by a process of genetically modifying said P-PHSC,
said process comprising: harvesting P-PHSC; after said harvesting,
culturing said harvested P-PHSC in a culture medium allowing for
proliferation of said P-PHSC; and after said culturing, introducing
a recombinant adeno-associated virus (rAAV)-vector into said
cultured P-PHSC to genetically modify said cultured P-PHSC.
2. The P-PHSC of claim 1 wherein said rAAV-vector comprises a
sequence encoding a protein of interest flanked by AAV inverted
terminal repeats (ITRs).
3. The P-PHSC of claim 1 wherein said rAAV-vector does not comprise
a promoter.
4. The P-PHSC of claim 1 wherein said rAAV-vector comprises a
promoter not derived from B19 parvovirus.
5. The P-PHSC of claim 4 wherein said rAAV-vector comprises a
functional part of the .beta.-globin promoter or a functional
analog thereof.
6. The P-PHSC of claim 4 wherein said rAAV-vector comprises a
herpes simplex virus thymidine kinase promoter or a functional
analog thereof.
7. The P-PHSC of claim 4 wherein said rAAV-vector comprises a
.DELTA.Mo+PyF101 Long Terminal Repeat promoter or a functional
analog thereof.
8. A cell derived from the P-PHSC of claim 1.
9. A cell derived from the P-PHSC of claim 2.
10. A cell derived from the P-PHSC of claim 3.
11. A cell derived from the P-PHSC of claim 4.
12. A cell derived from the P-PHSC of claim 5.
13. A cell derived from the P-PHSC of claim 6.
14. A cell derived from the P-PHSC of claim 7.
15. A method of potentiating transduction of P-PHSC with an
rAAV-vector comprising: harvesting P-PHSC; after said harvesting,
culturing said harvested P-PHSC in a culture medium allowing for
proliferation of said P-PHSC, said culture medium comprising a
hemopoietic growth factor; and after said culturing, introducing an
rAAV-vector into said cultured P-PHSC to genetically modify said
cultured P-PHSC.
16. The method of claim 15 wherein said hemopoietic growth factor
comprises interleukin-3 or a functional analog or fragment thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/326,032, filed Jun. 4, 1999, (U.S. Pat. No. ______) pending,
which claims priority under 35 U.S.C. .sctn..sctn. 119, 120 &
365 from, and is a continuation of, International Application No.
PCT/NL97/00631, filed on Nov. 19, 1997, designating the United
States of America. This application further claims benefit under 35
U.S.C. .sctn. 119 to EPO patent application 96203444.3 filed Dec.
5, 1996.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of gene therapy
and, more particularly, relates to DNA molecules derived from
adeno-associated virus (AAV) for the genetic modification of
primate hemopoietic stem cells.
BACKGROUND OF THE INVENTION
[0003] Genetic modification of pluripotent hemopoietic stem cells
from primates (P-PHSC) has been an elusive goal for many years.
Retrovirus vectors have been used in the past with limited success
(1). Though retroviral vector technology is still improving,
progress in increasing the transduction of P-PHSC is slow. This is
due to the fact that a solution is not straightforward and that the
P-PHSC cannot be identified by a rapid in vitro culture method (1).
Though culture of hemopoietic progenitor cells is possible, the in
vitro transduction levels of these cells do not reflect
transduction of P-PHSC that in vivo can grow out to give long term
reconstitution in multi-hemopoietic lineages (1,2,3). Although
long-term in vitro culture assays, such as the so-called LTC-IC
assay, have long been considered relevant assays for P-PHSC, it is
now generally accepted that only a very minor sub-population of the
cells identified in long-term in vitro culture assays are P-PHSC.
Therefore, genetic modification of long-term in vitro cultured
cells, even very efficient genetic modification, does not provide
any relevant information on genetic modification of P-PHSC.
Furthermore, although increasing knowledge is being gathered on the
expression of cell surface markers on P-PHSC, P-PHSC can also not
be identified by their phenotype. P-PHSC are known to express the
CD34 molecule and to be negative for many other hemopoietic cell
surface markers, but even the purest P-PHSC population that can
currently be phenotypically characterized contains only a few
P-PHSC. Due to this, transduction has to be evaluated by laborious
and lengthy in vivo studies using a bone marrow transplantation
setting where the stem cells in the bone marrow were transduced ex
vivo and subsequently transplanted back into monkey or human.
Transduction of P-PHSC is verified by the long term persistence of
genetically modified hemopoietic cells. Currently, the most
efficient method for the transduction of P-PHSC is by means of
retroviral vectors. Using such vectors, it is possible to transduce
approximately up to 0.01-0.1% of the P-PHSC (3,4,5,6,7). The
limitation of retroviral transduction is most likely due to a
restricted expression of the retrovirus receptor on P-PHSC,
combined with the fact that P-PHSC are usually not in cell cycle,
whereas retroviral vectors do not efficiently transduce
non-dividing cells (8,9,10,11).
[0004] A number of methods have been devised to improve the P-PHSC
transduction by retroviral vectors, such as pseudotyping
retroviruses using VSV (Vesicular Stomatitis Virus) envelope
protein or GALV (Gibbon Ape Leukemia Virus) envelope proteins to
target different and possibly more abundantly present receptors on
the cell membrane. Other strategies were directed toward improving
the number of cycling P-PHSC in the transplant. To date, this did
not result in a significant improvement of P-PHSC transduction.
[0005] In contrast to P-PHSC, murine PHSC are very easily
transduced by the current generation of retroviral vectors. This
observation, made in experiments using retroviral vectors, shows
that successful gene transfer into murine PHSC is by no means
indicative for successful gene transfer into P-PHSC. One can think
of a number of different possible reasons for this observation. We
hypothesized that it is theoretically not optimal to use a vector
system that has evolved in murine animals for humans. Though the
cellular processes involved in the murine retrovirus life cycle are
conserved between murine mammals and primates, it is very well
possible that the evolutionary divergence of the species resulted
in structural differences in the related proteins that affect the
functional efficiency of the murine virus proteins in human cells
and, thus, affect the transduction process. To avoid these
problems, we turned to a different vector system based on the human
adeno-associated virus (AAV).
[0006] AAV is a human virus of the parvovirus family. The AAV
genome is encapsidated as a linear single-stranded DNA molecule of
approximately 5 kb. Both the plus and the minus strand are
infectious and are packaged into virions (12,13). Efficient AAV
replication does not occur unless the cell is also infected by
adenovirus or herpes virus. In the absence of helper virus, AAV
establishes a latent infection in which its genome is integrated
into the cellular chromosomal DNA. The AAV genome contains two
large open reading frames. The left half of the genome encodes
regulatory proteins, termed REP proteins, that govern replication
of AAV-DNA during a lytic infection. The right half encodes the
virus structural proteins VP1, VP2, and VP3 that together form the
capsid of the virus. The protein coding region is flanked by
inverted terminal repeats (ITRs) of 145 bp each, which appear to
contain all the cis-acting sequences required for virus
replication, encapsidation and integration into the host chromosome
(14,15).
[0007] In an AAV-vector, the entire protein-coding domain (.+-.4.3
kb) can be replaced by the gene(s) of interest, leaving only the
flanking ITRs intact. Such vectors are packaged into virions by
supplying the AAV-proteins in trans. This can be achieved by a
number of different methods, one of them encompassing a
transfection into adenovirus infected cells of a vector plasmid
carrying a sequence of interest flanked by two ITRs and a packaging
plasmid carrying the in trans required AAV protein coding domains
rep and cap (15,16,17,18,19). Due to the stability of the
AAV-virion, the adenovirus contamination can be cleared from the
virus preparation by heat inactivation (1 hr, 56.degree. C). In
initial studies, virus preparations were contaminated with
wild-type AAV, presumably due to recombination events between the
vector and the helper construct (16,17,18,19). Currently, wild-type
AAV-free recombinant AAV stocks can be generated by using packaging
constructs that do not contain any sequence homology with the
vector (15).
[0008] Several characteristics distinguish AAV-vectors from the
classical retroviral vectors (See, e.g., Table 1). AAV is a DNA
virus, which means that the gene of interest, within the
size-constraints of AAV, can be inserted as a genomic clone (20,
21). Some genes, most notably the human .beta.-globin gene, require
the presence of introns for efficient expression of the gene (22).
Genomic clones of genes cannot be incorporated easily in retroviral
vectors, as these will splice out the introns during the RNA-stage
of their life-cycle (23).
[0009] In human target cells, wild-type AAV integrates,
preferentially, into a discrete region (19q13.3-qter) of chromosome
19 (24,25,26). This activity might correlate with rep-gene
expression in the target cell, since it was found that the large
REP-proteins bind to the human integration site in vitro (27).
AAV-vectors do integrate with high efficiency into the host
chromosomal DNA; however, thus far, they do not share the
integration site specificity of wild-type AAV (20). Site-specific
integration would be of great importance since it reduces the risks
of transformation of the target cell through insertional
mutagenesis. Wild-type AAV is, thus far, not associated with human
disease. Evidence is accumulating that AAV infection of a cell,
indeed, forms an extra barrier against its malignant transformation
(reviewed in (28)). In contrast to retroviral vectors where, due to
the extended packaging signal, parts of the gag-region need to be
present in the vector, the entire protein coding domain of AAV can
be deleted and replaced by the sequences of interest, thus totally
avoiding any immunogenicity problem associated with viral protein
expression in transduced target cells. One drawback of AAV-vectors
is that they are derived from a human virus. Thus, patients treated
with an AAV-vector might become exposed to wild-type AAV that, in
the presence of a helper virus such as adeno-virus or herpes
simplex virus, can supply the virus replication and packaging
proteins in trans and thus induce spread of the recombinant
AAV-virus into the environment. This is a feature not shared by the
currently used MuLV-derived retroviral vectors; wild-type MuLV's do
not normally cause infections in humans. The risk of recombinant
AAV spread into the environment must, however, not be overestimated
since it requires the presence of wild-type AAV and a helper virus.
This is not a frequently occurring situation. In addition, during
the integration process of AAV-vectors, often the ITRs undergo some
form of recombination leading to loss of function (15). Such
proviruses cannot be rescued and, thus, provide an additional
safety level of these vectors.
[0010] The first AAV-vectors were made by replacing part of the
AAV-coding region with either the Chloramphenicol Acetyltransferase
(CAT) or the neo.sup.R gene (16,17). All of these vectors retained
either a functional rep- or a functional cap-coding region.
Recombinant virus was generated by co-transfection with a plasmid
containing a complete AAV-genome. The recombinant AAV-CAT virus
conferred Chloramphenicol Acetyltransferase activity to 293 cells
(16), whereas the recombinant neo.sup.R virus conferred
G418-resistance to Human Detroit 6 cells, KB-cells and mouse
L-cells (17).
[0011] Currently, AAV-vectors are made that are totally devoid of
AAV-protein coding sequences. Typically, virus is made from these
vectors by complementation with a plasmid carrying the AAV-protein
coding region but no ITR-sequences (15).
[0012] AAV-vector technology is under development for a number of
different therapeutic purposes and target tissues. The as yet most
developed system is, perhaps, AAV-vector mediated gene transfer to
lung cells (29,30). AAV-vectors carrying the neo.sup.R gene or the
CAT gene were transferred and expressed efficiently in airway
epithelial cells (29). An AAV-vector carrying sequences 486-4629 of
the human Cystic Fibrosis Transmembrane conductance Regulator
(CFTR) gene fused to a synthetic oligonucleotide supplying the
translation start site, was capable of complementing Cystic
Fibrosis (CF) in vitro (31). In addition, stable gene transfer and
expression was reported following infection of primary CF nasal
polyp cells and after in vivo delivery of the AAV-CFTR vector to
one lobe of the rabbit lung (30). In vivo, the vector DNA could be
detected in 50% of the nuclei at 3 months post-administration.
Although the prevalence of the vector decreased after this time
point, .+-.5% of the nuclei still were positive at the six months
time point (30). The presence of the vector correlated well with
expression of RNA and recombinant protein, which were still
detectable at the six months follow up (30).
[0013] AAV-vector mediated gene transfer into murine hemopoietic
cells was demonstrated by the conferral of G418 resistance to
murine in vitro colony forming units (CFU) following infection with
a recombinant AAV-vector carrying the neo.sup.R-gene (32,33). The
presence of the vector in the progeny of CFU-GM (colony forming
units-Granulocyte Macrophage) and BFU-E (burst forming
units-Erythrocyte) was verified by means of PCR (Polymerase Chain
Reaction). The efficiency of gene transfer varied between 0.5% and
15% (33). Efficient gene delivery (up to 80%) into human
hemopoietic progenitors and human CD34.sup.+ cells with
AAV-neo.sup.R vectors has also been reported (34,35,36,37). These
studies demonstrated that recombinant AAV vectors were able to
deliver their DNA to the nucleus of the hemopoietic progenitor
cells that can be cultured in vitro. Though delivery of the vector
DNA to the nucleus of cells demonstrates the presence of a
functional virus receptor on the surface of the target cells,
delivery of recombinant AAV to the nucleus of cells is not directly
related to the integration of that DNA into the host cell genome
(discussed later and presented in Table 2). Recombinant AAV DNA
present as an episome in the cells is known to refrain from
integration into the host cell genome in non-dividing tissue
culture cells (38). Integration of recombinant AAV in CD34.sup.+
cells and in vitro growing colonies (CFU-C) was demonstrated in
1996 by Fischer-Adams et al. (59). Stable transduction of P-PHSC is
neither taught nor suggested in any of these prior art documents,
however. None of the above mentioned studies discloses delivery and
integration of recombinant AAV to P-PHSC, the only relevant
hemopoietic cell type for long term persistence of transduced cells
in vivo.
[0014] We are developing recombinant AAV gene transfer into P-PHSC
for the treatment of .beta.-thalassemia and Sickle cell anemia.
Both diseases severely affect the function of erythrocytes in these
patients. .beta.-thalassemic erythrocytes contain insufficient
.beta.-globin chains, whereas mutant .beta.-globin chains are made
in sickle cell anemia (for review, See (39)). Both diseases
severely affect erythrocyte function, which can be alleviated by
persistent .gamma.-globin gene expression in the adult patient, in
which case fetal hemoglobin is formed (40). Both inherited diseases
are recessive in nature, which indicates that one functional intact
copy of the adult .beta.-globin gene is sufficient to ameliorate
the phenotype.
[0015] Globin abnormalities were discarded as targets for gene
therapy attempts in the early days of gene therapy research. This
was largely due to the extremely complicated expression patterns of
globin-like genes (41). Globin-synthesis is highly regulated during
development and confined to cells of the erythroid lineage.
Furthermore, the expression of .alpha.- and .beta.-globin like
chains is regulated such that they are maintained at a 1 to 1 ratio
in the cell. Such careful control of gene expression is not easily
obtained. Expression vectors carrying the human .beta.-globin gene
with its promoter and local enhancer elements can direct erythroid
specific globin RNA expression (42). However, typically, the levels
are less than 1% of the endogenous globin RNA.
[0016] Recently, sequences 50-60 kb upstream of the .beta.-globin
gene were discovered that direct the high level, tissue specific,
copy number dependent, and position independent expression of the
.beta.-globin gene (43). This region, designated the Locus Control
Region (LCR), is characterized by four strong erythroid-specific
DNaseI hypersensitive sites (HS1-4) (44). Fine-mapping of the
active sequences in the LCR identified four fragments of .+-.400 bp
in length that each coincide with one HS site. Walsh et al
incorporated a marked .gamma.-globin gene and the core fragment of
HS2 together with the neo.sup.R gene into an AAV-vector (20).
Infected and G418-selected pools and clones of K562 cells produced
the marked .gamma.-globin RNA to 50-85%, compared to the normal
level expressed by one endogenous .gamma.-globin gene (20,45). A
drawback of this vector is that the .gamma.-globin gene and
promoter used in these studies are specific for expression in fetal
erythroid tissue and are, thus, not ideal for use as a therapeutic
agent in adult humans. Incorporation of .beta.-LCR sites 1, 2, 3,
and 4 in a vector containing the adult specific human p-globin gene
resulted in a very high regulated expression in MEL (murine
erythroleukemia) cells, the best in vitro marker cell line for
regulated erythroid expression in adult tissue (46). The present
invention describes the use of this and similar vectors in the
transduction of P-PHSC.
[0017] The term "infectious particles" is used herein to refer to
AAV particles that can deliver their packaged DNA to the nucleus of
cells and replicate in the presence of adenovirus and wild-type
AAV.
[0018] The term "transducing particles" is used herein to refer to
AAV particles that can deliver their packaged DNA to the nucleus of
target cells where the packaged DNA is released and integrates into
the chromosomal DNA of the target cells.
SUMMARY OF THE INVENTION
[0019] This invention provides a process of genetic modification of
pluripotent hemopoietic stem cells of primates (P-PHSC), comprising
introducing a nucleic acid molecule based on adeno-associated virus
(AAV), in particular a recombinant AAV, which is derived from human
AAV, into P-PHSC, preferably by transduction. The genome of the
recombinant AAV comprises a DNA sequence flanked by the inverted
terminal repeats (ITR) of AAV, or functional analogs or fragments
thereof. Normally and preferably, but not necessarily, said DNA
sequence will be a non-AAV DNA sequence, in particular a
therapeutic DNA sequence.
[0020] According to a preferred embodiment of the invention, the
DNA sequence comprises regulatory sequences functional in
hemopoietic cells (in particular hemopoietic stem cells) and, under
the control of said regulatory sequences, a sequence coding for a
protein or RNA with a therapeutic property when introduced into
hemopoietic (stem) cells. Preferred examples of the DNA sequence
comprise the coding sequence of such genes as the human lysosomal
glucocerebrosidase gene (E.C.3.2.1.45), a globin gene from the
human .beta.-globin gene cluster, a DNA sequence encoding an RNA or
protein with anti-viral activity, the .alpha.1-antitrypsin gene and
the human multidrug resistance gene I (MDRI).
[0021] In a particularly preferred embodiment, the DNA sequence
comprises the human .beta.-globin gene inclusive of at least one of
its introns or functional analogs thereof, under transcriptional
control of a functional part of the .beta.-globin promoter or
functional analogs thereof, and being operably linked to
erythroid-specific DNaseI hypersensitive sites from its Locus
Control Region (LCR), more particularly, the .beta.-LCR elements
HS4, HS3, and HS2 or functional analogs thereof.
[0022] The DNA sequence also may comprise a selectable marker gene
useful in hemopoietic stem cells, such as a neo.sup.R gene, under
transcriptional control of a herpes simplex virus (HSV) thymidine
kinase (tk) promoter or functional analogs thereof or a
.DELTA.Mo+PyF101 Long Terminal Repeat (LTR) promoter.
[0023] The P-PHSC may be obtained from primate bone marrow, cord
blood, or peripheral blood and, preferably, is human-derived. The
P-PHSC may be exposed in vitro to proliferation stimulating
compounds, such as interleukin 3 or a functional analog or fragment
thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention is based on the discovery that
AAV-derived vectors efficiently transduce primate pluripotent
hemopoietic stem cells. To date, AAV has not been reported to
transduce pluripotent hemopoietic stem cells of primates, and
AAV-derived vectors have not been shown to transduce hemopoietic
cells with in vivo repopulating ability. Also, it was reported that
primary cells are much less efficiently transduced by recombinant
AAV than are immortalized cell lines (47). In addition, it was
reported that orf6 from the adenovirus E4-region stimulates
transduction by recombinant AAV (48).
[0025] A surprising and novel aspect of the present invention is
that the rAAV-vector integrates with higher efficiency in cultured
P-PHSC than in non-cultured P-PHSC, even though most of the
cultured P-PHSC are not actively dividing at the time of infection.
This is surprising, since it has been established that recombinant
AAV integration in dividing cells occurs 200 times more efficiently
than in non-dividing cells (38). However, in non-cycling cells the
vector remains in the nucleus and retains its ability to integrate
when the cell is triggered into cycle (60). Therefore, a difference
in transducibility of cultured versus non-cultured cells is not
expected when only replication of the target cells is the enhancing
factor. We thus infer that culture and exposure to hemopoietic
growth factors such as interleukin-3 could in other ways potentiate
the transduction of P-PHSC with recombinant AAV.
[0026] In a gene therapy setting, it is undesirable to have
functionally active adenovirus present due to toxicity problems
caused by the virus directly or the immune system of the patient.
At the Keystone Symposium on Molecular and Cellular Biology, Taos,
N. Mex. Feb. 4-10, 1996, Prof. A. Nienhuis presented a paper
stating that they transduced rhesus monkey CD34.sup.+ cells and,
subsequently, autologously transplanted the infected cells (49).
Analysis of the peripheral blood cells circulating in blood with a
polymerase chain reaction specific for the recombinant AAV revealed
that cells carrying the recombinant AAV were only detected up until
7 days post transplantation (49), that is to say, P-PHSC were not
transduced by recombinant AAV in their experiment. Nonetheless, the
present invention demonstrates that an AAV-derived vector may be
used to deliver exogenous DNA efficiently to cells of the
hemopoietic system with long term repopulating ability.
[0027] The current perception of AAV-integration into the cellular
host chromosome is that the pre-integration complex is stable in
cells. Although integration occurs more efficiently in dividing
cells, the pre-integration complex is stable in non-dividing cells
and integrates when the cell is triggered to undergo cell cycling
(38,60). The primate-derived hemopoietic stem cells and committed
progenitor cells, upon autologous transplantation into an
irradiated recipient, are triggered into cycle to repopulate the
destroyed hemopoietic system. For this reason, it is generally
believed that the hemopoietic cells need not be triggered in vitro.
Therefore, protocols to transduce hemopoietic progenitor cells with
recombinant AAV do not involve culturing the cells in the presence
of hemopoietic growth factors. Although this reasoning is very
plausible with the current information, we devised experiments to
investigate the effect of in vitro culture of hemopoietic stem
cells and the in vitro stimulation with hemopoietic growth
factors.
[0028] As used herein, the term "recombinant AAV-vector" means a
DNA sequence flanked at each end by an AAV-ITR or functional
equivalent or part thereof. The recombinant AAV vector can be used
directly or can be packaged into a complex before use. As used
herein, the term "complex" is defined as a combination of two or
more components physically linked to each other through
hydrophobic, hydrophilic or electrostatic interactions or covalent
bonds, whereby at least one component of the complex is a
recombinant AAV molecule. Other components of the complex can
comprise, but are not limited to, one or a combination of
liposomes, calcium phosphate precipitate, polylysine, Adenovirus,
Adenovirus proteins, Rep78, Rep68, AAV capsids, or the AAV capsid
proteins VP1, VP2, or VP3. In a preferred embodiment, the complex
consists of the recombinant AAV vector and the AAV capsid proteins.
This complex can be, but is not limited to, in the form of an
intact virion or a particle where the recombinant AAV vector is
packaged inside an AAV capsid or functional analogs thereof.
[0029] As used herein, the term "functional analogs" refers to the
same activity in kind, but not in amount or degree, i.e. not
quantitatively.
[0030] When the recombinant AAV is packaged into AAV particles, the
size of the DNA sequence will be limited by the size constraints
for packaging into AAV particles which, with the current state of
the technology, is about 5 kb. The DNA fragment preferably does not
contain sequences functionally analogous to the terminal resolution
site in the AAV-ITR as this might interfere with the stability of
the recombinant vector. The DNA sequence can be any sequence with
therapeutic properties when introduced into hemopoietic stem cells,
but the DNA sequence preferably encodes one or more proteins or RNA
with therapeutic properties when expressed in hemopoietic cells.
Non-limiting examples of such sequences are the human .beta.-globin
gene operably linked to cis-acting sequences for erythroid specific
physiological expression, the human lysosomal glucocerebrosidase
gene (E.C.3.2.1.45), the .alpha.1-antitrypsin gene, a DNA sequence
encoding an RNA or protein with anti-viral activity or the
multidrug resistance gene I (MDRI). AAV-ITR sequences may be
obtained from AAV serotypes 1, 2, 3, 4 or 5. Alternatively, mutant
or recombinant ITR sequences can be used, which retain the
essential properties of the AAV-ITR prototype, examples of which
are described in Lefebvre et al. (50).
[0031] Packaging of recombinant AAV into AAV-virions can be
achieved using a variety of different methods. All methods are
based on bringing the necessary proteins and recombinant
AAV-containing DNA into an environment that supports the
replication and packaging of recombinant AAV. One method relies on
the transfection of adenovirus 5-infected human cells with a
plasmid carrying the recombinant AAV-DNA together with a plasmid
containing expression cassettes for the AAV-genes rep and cap. Upon
continued culture of the manipulated cells, recombinant AAV is
replicated and packaged. After three days, the cells are harvested
and the accumulated recombinant virions are released from the cells
(15-19). A variation on the packaging system described above is the
use of packaging cells that carry all or part of the relevant
sequences stably integrated in their genome (i.e. the recombinant
AAV vector, the rep-gene, the cap-gene, and the relevant protein
coding domains of the helper virus). When only partial packaging
cells are used, the missing packaging functions have to be supplied
externally via transfections of plasmids carrying the functions or
virus infection. The helper virus functions are required for
efficient packaging of recombinant AAV. For most applications, the
helper virus is inactivated or separated physically from the
recombinant AAV virions before using the recombinant AAV virions
for the transduction of cells (15-190). Recombinant AAV vectors can
be packaged by adding the recombinant AAV-DNA to protein extracts
or mixtures of protein extracts of cells that expressed all or part
of the relevant proteins for the replication and packaging of
recombinant AAV. When protein extracts are used from cells
expressing only some of the relevant proteins for packaging of
recombinant AAV, the missing proteins can be supplied externally in
purified form.
[0032] The rep-gene can be derived from AAV serotypes 1-5, or
functional analogues thereof, either obtained through non-essential
mutations in the rep-genes or through the isolation of genes with
similar capabilities, such as the Human Herpesvirus 6 AAV-2 rep
gene homologue (58).
[0033] The cap-gene can be derived from AAV serotypes 1-5, or
functional analogues thereof, obtained through non-essential
mutations in the cap-genes. Alternatively, the cap-gene sequences
can be altered through the replacement or addition of sequences
providing the produced virion new or altered target cell
specificities.
[0034] Recombinant AAV virions produced by the methods described
above can be purified and concentrated using biological, physical,
or chemical separation techniques such as, but not limited to,
antibody affinity purification, density gradient centrifugation, or
ion exchange chromatography. Alternatively, the virions produced
can be used in an unpurified form.
[0035] As used herein, pluripotent hemopoietic stem cells from
primates (P-PHSC) are functionally defined as cells from primates
with the capability to form and maintain an entire hemopoietic
system, ranging from mature T-cells, B-cells, macrophages, or
erythrocytes to new P-PHSC. P-PHSC display this capability in
unmanipulated primates or upon their autologous transplantation.
Sources of P-PHSC are the bone marrow, the peripheral blood, or the
cord blood. P-PHSC can be collected from unmanipulated primates or
from primates treated with compounds such as, but not limited to,
cytostatic drugs or hemopoietic growth factors to activate,
recruit, or otherwise potentiate the P-PHSC.
[0036] Transduction of P-PHSC is preferably performed ex vivo
following harvesting of the P-PHSC from a suitable source, and
after the transduction, the transduced cells are autologously
transplanted. In a preferred embodiment of the invention, the
P-PHSC are cultured during their ex vivo transduction, where it is
most preferred that during this culture the P-PHSC are stimulated
with at least one hemopoietic growth factor, such as interleukin-3.
Alternatively, P-PHSC transduction is performed in vivo when
suitable methods have been developed to target the recombinant AAV
vector in vivo to P-PHSC.
BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS
[0037] Table 1. Key properties of Adeno-associated virus vectors
and amphotropic retrovirus vectors.
[0038] Table 2. Characterization of recombinant AAV preparations
useful for the transduction of P-PHSC.
[0039] Table 3. Transduction of P-PHSC: culture and infection
conditions.
[0040] IP=Infectious Particles (titrated in RCA);
[0041] TP=Transducing Particles (titrated on MEL cells).
[0042] Table 4. Transduction of P-PHSC: Hemopoietic data.
[0043] FIG. 1A. Recombinant AAV-vectors useful for the transduction
of P-PHSC.
[0044] ITR=Adeno-associated virus inverted terminal repeat.
[0045] LCR=Core sequences from hypersensitive sites 4, 3, and 2
from the .beta.-globin locus control region.
[0046] -103=human .beta.-globin gene promoter fragment extending
-103 upstream of the transcription start site.
[0047] -265=human .beta.-globin gene promoter fragment extending
-265 upstream of the transcription start site.
[0048] .beta.-globin=human .beta.-globin gene with modified intron
2 (see text and 21).
[0049] Tkprom=Herpes Simplex Virus Thymidine kinase gene promoter
(approx. 500 bp NarI-BglII fragment).
[0050] NEO=BglII-SmaI fragment from E. coli Tn5 transposon.
[0051] pA=Polyadenylation signal from Herpes Simplex Virus
Thymidine Kinase gene (approx. 500 bp SmaI-NarI fragment).
[0052] .beta.*-globin=human .beta.-globin gene containing in the 5'
untranslated region three point mutations that generate two
restriction enzyme sites (see FIG. 1B).
[0053] .DELTA.Mo+PyF101=a Moloney murine leukemia virus long
terminal repeat fragment in which the Moloney enhancer is replaced
by an enhancer from a mutant polyoma virus that was selected to
grow on embryonal carcinoma cells (2,51,52,53).
[0054] FIG. 1B. Nucleotide sequence of the 5' untranslated region
(UTR) of the normal (.beta.) and the marked (.beta.*) human
.beta.-globin gene.
[0055] FIG. 2. Detection of recombinant AAV in rhesus monkey
peripheral blood cells. Blood cells were collected as described in
the specification. Peripheral blood mononuclear cells (WBC) were
separated from the granulocytes (Gran) and a neo-specific nested
PCR was performed on the DNA of both cell types. DNA from the
nested PCR was analyzed on agarose gels and compared to positive
and negative control samples. The sensitivity of the nested PCR was
such that approximately one recombinant AAV-vector could be
detected in a background of 10.sup.5 negative cells. (+) indicates
the presence of a neo-specific band and (-) the absence of a
neo-specific band in the agarose gel.
[0056] FIG. 3. Graphic representation of direct and nested
neo-specific PCR data from monkeys BB94 and A94 (FIG. 3a) and
monkeys 9128 en 9170 (FIG. 3b). The data on the latter two monkeys
shown in FIG. 2 are included in FIG. 3 as well. For clarity,
negative PCR-results were not included in the graphs. Closed
circles (PBMC) and closed squares (Granulocytes) indicate the
time-points after transplantation at which the vector was detected.
Arrows in FIG. 3b indicate the time-points at which docetaxel
(Taxotere) was administered.
[0057] FIG. 4 Detection of neo-specific sequences in hemopoietic
cells from rh BB94 at 16 months post transplantation. BM (bone
marrow), PBMC (peripheral blood mononuclear cells), Gran
(granulocytes).
[0058] FIG. 5 Detection of vector specific globin sequences in
rhesus monkey peripheral blood cells (samples from 2 months (A94)
and 6 months (BB94) post-transplantation). With this PCR, the two
vectors IG-CFT and IG-CFT* are discriminated since the size of the
IG-CFT* fragment is approximately 150 bp longer than the fragment
specific for IG-CFT.
EXAMPLE 1
Ligation of Recombinant AAV Vectors Containing the Human
.beta.-globin Gene and/or the neo.sup.R Gene
[0059] In order to determine whether recombinant AAV could
transduce P-PHSC, it was necessary to generate appropriate vectors.
We generated three different recombinant AAV-vectors, which are
schematically represented in FIG. 1A. The ligation of the vector
IG-CFT containing a human .beta.3-globin gene together with
sequences from the .beta.-globin locus control region and the
neo.sup.R-gene is described in (21). IG-CFT.sup..times. differs
from IG-CFT in the size of the human .beta.-globin promoter and in
the presence of three point mutations in the 5' untranslated region
(UTR) of the human .beta.-globin gene. In IG-CFT*, the promoter
driving .beta.-globin expression extends 265 bp upstream of the
transcription start site instead of the 103 bp in IG-CFT. In
IG-CFT*, three point mutations in the 5' UTR of the human
.beta.-globin gene created two new restriction sites, one for XbaI
and one for HindIII (See FIG. 1B).
[0060] IG-.DELTA.MoNeo (depicted in FIG. 1A) contains the
recombinant AAV-backbone (XbaI-fragment) from pSub201(15), the
NheI-SmaI promoter-fragment from the .DELTA.Mo+PyF101 LTR (53), the
BglII-SmaI fragment from the Tn5-derived neo.sup.R-gene followed by
the SmaI-NarI polyadenylation signal from Herpes Simplex Virus
(HSV) Thymidine Kinase (TK) gene (54). The elements were linked
together using the polylinker from pBluescript SK.sup.+
(Stratagene).
EXAMPLE 2
Production of Recombinant AAV from IG-CFT, IG-CFT* and
IG-.DELTA.MoNeo
[0061] The 293 cell line (55), which is a human embryonic kidney
cell line transformed with Ad5 DNA, the A549 cell line, which is a
human bronchial carcinoma cell line, and the C88 cell line (56),
which is a murine erythroleukemia (MEL) cell line, were maintained
in DMEM (GIBCO-BRL) containing 10% Fetal Calf Serum (FCS), 100
.mu.g/ml streptomycin, and 100 U/ml penicillin. Recombinant AAV was
produced by transfecting a recombinant AAV packaging plasmid and a
vector plasmid into approx. 90% confluent permissive 293 cells. The
cells were made permissive for AAV-replication by transfecting them
with a plasmid capable of expressing all the relevant early genes
from adenovirus but not the late genes or by infecting them with
adenovirus ts149 with a multiplicity of infection of 20. The
packaging plasmid was either pAAV/Ad (15) or pIM45, which contains
sequences 146 to 4493 from wild-type AAV2 in the polylinker of
pBluescript. The ratio of vector plasmid DNA to packaging plasmid
DNA was 1:10 to accommodate the fact that the recombinant AAV
vector replicates upon expression from the packaging plasmid,
whereas the packaging plasmid does not replicate. For crude virus
stocks, the cells were harvested in their own culture medium after
two to three days and subjected to three freeze/thaw cycles. The
latter was performed by intermittent freezing and thawing in liquid
nitrogen and a 37 .degree. C. water bath. Cell debris was
subsequently pelleted (10 min, 200 g) and the supernatant was
incubated at 56.degree. C. for 1 hour to inactivate residual
adenovirus. Concentrated high titer recombinant AAV stocks were
prepared by harvesting the cells in their own culture medium and
washing in PBS (max. 10.sup.7 cells/ml). The virus was released
from the cells by 3 freeze/thaw cycles and/or 30 sonication pulses
of 1 second on ice to prevent warming. Cell debris was spun down
and the supernatant was made a density of 1.4 by adding solid CsCl.
After o/n centrifugation (50.000 r.p.m., 20.degree. C., using a vti
T165.1 rotor in a Beckman ultracentrifuge), fractions were
collected and recombinant AAV was determined. Fractions containing
recombinant AAV were pooled and further concentrated in a centricon
concentrator (Amicon) according to manufacturer's specifications.
After concentration, the medium containing the virus was changed by
two successive washes in the centricon concentrator using Optimem
culture medium (GIBCO-BRL).
EXAMPLE 3
Characterization of Recombinant AAV Preparations
[0062] To determine the effect of the different methods of virus
preparation and the different processing steps on the quality of
the various recombinant AAV-batches, we characterized them for 5
parameters: 1) the capacity to deliver the desired DNA to the
nucleus of the target cell by means of a replication center assay
(RCA) described below, 2) the capacity to stably transduce cells
and express the neo.sup.R-gene by means of a limiting dilution on
MEL cells followed by G418 selection, 3) the wild-type AAV titer in
the batches by a RCA without added wild-type AAV, 4) the amount of
replication proficient adenovirus in each preparation, and 5) the
concentration of CsCl in the recombinant AAV preparations that were
purified using CsCl gradients (See Table 2).
[0063] Replication Center Assay
[0064] The replication center assay (RCA) takes advantage of the
fact that in a lytic infection of AAV up to 10.sup.6 AAV, genomes
are produced inside a cell. This amount of DNA is sufficient for
the radioactive detection of infected cells. To accomplish this,
293 cells were seeded in a flat bottom 96 wells plate such that
they reached near confluence the following day. For a titration of
recombinant AAV, the cells were infected with dilutions of
recombinant virus stock, adenovirus ts 149 (M.O.I. 20) and
wild-type AAV-2 (M.O.I. 2). For a titration of the wild type AAV,
the cells were infected with dilutions of recombinant virus stock
and adenovirus ts 149 (M.O.I. 20). The cells were subsequently
incubated at 39.degree. C. The next day, after 24 hours, the medium
was replaced by ice-cold PBS containing 5 mM EDTA. After 5 to 20
minutes on ice, a single cell suspension was made by rigorous
pipetting. The cells were diluted in 5 ml PBS and sucked onto
hybond N.sup.+ filter circles (pore size 0.22 .mu.m) of 3.6 cm
diameter. Filters were incubated for 5 minutes in denaturation
solution (0.4 M NaOH; 0.6 M NaCl) and 5 min in renaturation buffer
(1.5 M NaCl; 1 M Tris-HCl, pH 7). Filters were washed and stored in
5.times.SSPE until hybridization. Filters were hybridized with a
recombinant AAV specific probe for the determination of the
recombinant AAV titer and hybridized with a wild type AAV specific
probe for the determination of the wild-type AAV titer.
[0065] MEL-cell Transduction
[0066] 1.5.times.10.sup.5 MEL cells were seeded in 2 ml culture
medium per well (24 wells plate, Falcon) and the appropriate
dilution of recombinant AAV virus was added. The cells were
collected the next day and reseeded in 30 ml culture medium in a 75
cm.sup.2 flask (Falcon). After three days, the medium was replaced
by selection medium by spinning down the cells (200 g, rt) and
resuspending the cells in fresh medium containing 1 mg/ml (dry
weight) G418 (Gibco). Medium was replaced every three to four days.
After fourteen days, the cultures were scored. When the cells had
grown to confluency, the cultures were scored positive since the
specific virus dilution contained recombinant AAV capable of stably
transducing MEL cells. Specific virus dilutions were scored
negative when, after fourteen days, confluency had not been
reached.
[0067] Adenovirus was determined by serial dilutions of the AAV
virus stock on A549 (human bronchial carcinoma) cells. Dilutions
were scored positive when cytopathic effect was visible after 6
days. Wild-type Adenovirus 5 stocks with a known titer were used as
positive controls. CsCl concentrations in the AAV preparations were
determined by flame photometry.
[0068] A summary of the characterization is given in Table 2. The
infectious particle (IP) concentration, that is to say, the
capacity to deliver recombinant AAV-DNA to the nucleus of target
cells, determined in the RCA varied considerably among the
different batches. Also, the transducing particle (TP)
concentration and the amount of wild-type AAV contamination varied
considerably. Three batches had an IP to TP ratio of 10.sup.4, the
248 crude batch had a much lower ratio of 200.
EXAMPLE 4
Transduction and Autologous Transplantation of Rhesus Monkey Bone
Marrow
[0069] Animal Care and Transplantation
[0070] The animals used for transplantation were 3-5 kg rhesus
monkeys (Macaca mulatta), bred at the Biomedical Primate Research
Centre (BPRC), Rijswijk, The Netherlands. Three weeks before
transplantation, the animals were transferred to a laminar flow
unit and selectively decontaminated in the digestive tract by
treatment with metronidazole (40 mg/kg/day), during 5 days,
followed by daily oral administration of ciprofloxacin (6.5
mg/kg/day), polymixin B (10 mg/kg/day) and nystatin (40
kU/monkey/day). A94 and BB94 received one administration of
ivermectine 200 .mu.g/kg anti-worm treatment approximately two
weeks prior to transplantation. The monkeys were kept under barrier
nursing and antimicrobial treatment until leukocyte counts exceeded
a value of 1.times.10.sup.9/liter. The day before transplantation,
the monkeys received 5 Gy total-body X-ray irradiation. For this
purpose, the animals were placed in a cylindrical polycarbonate
cage which rotated 6 rpm around its vertical axis during
irradiation from two opposing beams (physical parameters: 300 kV, 7
mA, 0.26 Gy/min dose rate, 0.80 m average focus-to-skin distance).
Bone-marrow grafts were infused into a peripheral vein in a volume
of 7.5 ml 0.9% NaCl. Supportive care after transplantation included
blood transfusions of 15 Gray-irradiated thrombocytes when
thrombocyte counts were below 40.times.10.sup.9/liter, subcutaneous
fluid upon indication and red blood cell transfusions when
hematocrit levels dropped below 0.2 l/l. Monkey 9128 was
administered daily Baytrill s.c. for 2 weeks, 9 months after
transplantation, as treatment of a Salmonella infection. Monkeys
BB94 and A94 were treated for Streptococci sepsis and received
cefamandolnafaat 50 mg/kg/day and tobramycine 3 mg/kg/day. A94 was
additionally treated for Streptococci sepsis with amoxiline 9
mg/kg/day, clavulanic acid 2.5 mg/kg/day and ceftriaxone 50
mg/kg/day and with Amphotericin B 8 mg/kg/day for a yeast
infection. Selective decontamination was stopped a few days after
hemopoietic repopulation of the monkeys. Sepsis treatment was
stopped 4 days after the body temperature had returned to normal
and serum cultures were found to be sterile. Docetaxel
(Taxotere.RTM.) treatment was given to monkeys rh9128 and rh9170 at
indicated times (FIG. 3) at a dose of 50 mg/m.sup.2. In monkey
rh9128, around 14 months post transplantation 4 docetaxel doses
were given of 10 mg/m.sup.2. The appropriate amount of docetaxel
was diluted in 50 ml PBS-Glucose (NPBI, The Netherlands) and was
administered by IV injection at a rate of 1 ml/min.
[0071] Bone Marrow Processing and Transduction
[0072] 40 ml of bone marrow aspirate was obtained by puncturing
both femoral shafts under total anesthesia. Bone marrow cells were
collected in Hanks' basic salt solution containing heparin at 100
units per ml and deoxyribonuclease-I and subjected to
Ficoll-Hypaque (Sigma) centrifugation. CD34.sup.+ selection was
performed using a small-scale CEPRATE LC column (CellPro, Bothell,
Wash.). From 5.times.10.sup.4 to 50.times.10.sup.4 cells were
incubated at 4.degree. C. for 30 minutes in 0.1 ml PBS and 1%
bovine serum albumin (BSA) with 5 ml of a phycoerythrin-conjugated
anti-CD34 antibody (563.F) or unconjugated anti-CD34 antibody
(566). Cells incubated with the antibody 566 were washed (PBS, 0.1*
BSA) and further incubated with PerCP conjugated Rabbit anti-Mouse
IgG1 (Becton-Dickinson, Cat no. 340272). After washing, cells were
acquired on a FACSort (Becton-Dickinson) flow cytometer. Cells were
analyzed with the Lysis II software program. The percentage of
CD34.sup.+ cells was calculated as the ratio of CD34.sup.+ cells to
total number of cells and multiplied by 100. For rhesus monkeys
9128 and 9170, the enriched CD34.sup.+ cells were immediately
processed for transduction. For rhesus monkeys A94 and BB94, the
enriched CD34.sup.+ cells were split into two equal fractions and
stored in liquid nitrogen.
[0073] Transduction of CD34.sup.+ cells was done as described
below. A summary of the experimental conditions is given in Table
3.
[0074] Rhesus monkey 9128 and 9170: Four days prior to
transplantation the CD34.sup.+ enriched cells were split in two
equal fractions and cultured at a density of 10.sup.6 cells per ml
in low density BMC culture medium supplemented with recombinant
rhesus monkey interleukin-3 (RhIL-3; Burger et al., 1990) as
described in (57). On day 2 and day 3, one fraction of cultured
CD34.sup.+ cells was exposed to the crude recombinant AAV
preparation of IG-CFT and the other fraction was exposed to a crude
recombinant AAV-preparation of IG-.DELTA.MoNeo by adding an equal
volume of virus preparation to the medium of the cultured
CD34.sup.+ cells. After three to five hours, the cells were
collected by centrifugation (7 min, 200 g) and resuspended into
fresh RhIL-3 supplemented low density BMC culture medium in the
same volume as the culture was started in. On day four, the cells
were collected by centrifugation (7 min, 200 g) and resuspended in
an equal volume of 0.9% NaCl and separately transplanted into
autologous rhesus monkeys by IV injection.
[0075] Rhesus monkey A94 and BB94: Four days prior to
transplantation, one fraction of the frozen CD34.sup.+ enriched
cells was thawed and subsequently washed with Hanks Balanced Salt
solution. Live cells were collected by Ficoll-Hypaque (Sigma)
centrifugation and cultured at a density of 10.sup.6 cells per ml
in Iscove's modified Eagles medium (IMDM, Gibco-BRL) supplemented
with Fetal Calf's Serum (FCS, 10%) and recombinant rhesus monkey
interleukin-3 (RhIL-3; Burger et al., 1990). On day 2 and day 3,
cells were collected by centrifugation (7 min, 200 g) and
resuspended in 10 to 200 .mu.l of IMDM+10% FCS and RhIL-3 and
subsequently exposed to a purified recombinant AAV preparation of
IG-CFT (Monkey A94) or IG-CFT* (Monkey BB94). After two hours, the
cells were washed with IMDM+10% FCS and reseeded in IMDM+10% FCS
and Rh-IL-3. At day four, the cells were collected by
centrifugation and suspended in 0.9% NaCl. Also, on day four, the
other fraction of CD34.sup.+ cells was thawed and washed with Hanks
Balanced Salt solution. Live cells were collected by Ficoll-Hypaque
(Sigma) centrifugation, resuspended in 10 to 200 .mu.l of IMDM+10%
FCS and RhIL-3 and subsequently exposed to a purified recombinant
AAV-preparation of IG-CFT (Monkey BB94) or IG-CFT* (Monkey A94).
After two hours, the cells were collected by centrifugation and
suspended in 0.9% NaCl. After collection in NaCl (0.9%), the cells
were separately transplanted into autologous irradiated rhesus
monkeys by IV injection.
[0076] Parameter Evaluation
[0077] Daily observation of clinical signs. Weekly complete
physical examination and determination of body weight. Blood
chemistry analysis was performed before and after X-ray
irradiation. Hematology was performed weekly. Bone marrow samples
were punctured from the femoral shafts under total anesthesia.
Heparine blood samples were taken weekly for PCR analysis. PBMC and
granulocytes were isolated from peripheral blood samples, as
described previously by Ficoll-Hypaque centrifugation (Van
Beusechem et al., 1992). Circulating T- and B-cells were purified
from PBMC by sorting CD2 and CD20 positive cells, respectively.
FTIC labeled CD2 (clone S 5.2; Becton-Dickinson, California) or
CD20 (clone L27; Becton-Dickinson, California) antibodies were
incubated with PBMC according to the manufacturers protocols.
Labeled cells were separated using the MACS.RTM. column and
anti-FITC beads (Miltenyi, Germany) according to the manufacturers
protocol. Re-analyses of the sorted cells on FACS.RTM.
(Becton-Dickinson, USA) showed that the sorted cells were more then
95% pure populations.
[0078] Colony-forming Cell (CFU-C) Assay
[0079] Rh9128 and Rh9170 hemopoietic cells were plated in duplicate
at 5.times.10.sup.3/ml (CD34.sup.+ selected) or 1.times.10.sup.5/ml
(post-Ficoll) in 1 ml methylcellulose medium, as described in (57),
supplemented with 30 ng/ml rhIL-3 and 25 ng/ml GM-CSF. Rh A94 and
BB94 hemopoietic cells were seeded for colony formation in
methylcellulose medium containing 50 ng/ml SCF, 10 ng/ml GM-CSF, 10
ng/ml IL-3 and 3 U/ml Epo (MethoCult GF H4434, StemCell
Technologies Inc, Vancouver, Canada).
[0080] Polymerase Chain Reaction
[0081] For cell lysis, pellets were incubated (10.sup.7 cells/ml)
in nonionic detergent lysis buffer (0.5% NP40, 0.5% Tween 20, 10 mM
Tris pH 8.3, 50 mM KCl, 0.01% gelatin, 2.5 mM MgCl.sub.2)
containing proteinase K (60 mg/ml) at 56.degree. C. for 1 hour.
Lysates were then heated at 95.degree. C. for 10 min to inactivate
the proteinase K. Two different PCR detections were performed. One
was a nested neo.sup.R-specific PCR and one was a .beta.-globin
specific PCR. The protocol for the neo.sup.R-specific PCR will be
described first. The first amplification was performed on 10 .mu.l
lysates in a total volume of 50 .mu.l with 2 U of SuperTaq
polymerase (HT Biotechnology, Cambridge, England) in a reaction mix
(final concentration: 200 mM each of 2'-deoxyadenosine-5'-tr-
iphosphate, 2'-deoxycytidine-5'-triphosphate,
2'-deoxyguanosine-5'-triphos- phate,
2'-deoxythymidine-5'-triphosphate (Pharmacia, Roosendaal, The
Netherlands), 0.2 .mu.M each of 5' neo-1 and the antisense primer
3' neo-2 and the reaction buffer supplied by the manufacturer (HT
Biotechnology, Cambridge, England). The nested amplification was
performed on 5 .mu.l of the first reaction in a total volume of 50
.mu.l with 2 U of SuperTaq polymerase (HT Biotechnology, Cambridge,
England) in a reaction mix (final concentration: 200 mM each of
2'-deoxyadenosine-5'-triphosphate,
2'-deoxycytidine-5'-triphosphate,
2'-deoxyguanosine-5'-triphosphate,
2'-deoxythymidine-5'-triphosphate (Pharmacia, Roosendaal, The
Netherlands), 0.2 .mu.M each of 5' neo-2 and the antisense primer
3' neo-1 and the reaction buffer supplied by the manufacturer (HT
Biotechnology, Cambridge, England). Primers were chosen to
selectively amplify the neo.sup.R gene.
[0082] The primer sequences are:
1 5' neo-1: 5'-GGGGTACCGCCGCCGCCACCATGATTGAACAAGATGGATTGC-3' (SEQ.
ID. NO. 1) 5' neo-2: 5'-TTCTCCGGCCGCTTGGGTGG-3' (SEQ. ID. NO. 2) 3'
neo-1: 5'-GGCAGGAGCAAGGTGAGATG-3' (SEQ. ID. NO. 3) 3' neo-2:
5'-CCATGATGGATACTTTCTCG-3' (SEQ. ID. NO. 4)
[0083] Amplification conditions were the same for the first and the
nested amplification and were performed in a TRIO thermocycler
(Biometra, Gottingen, Germany) temperature cycling apparatus. The
conditions chosen were: 95.degree. C. for 5 minutes, then 30 cycles
of 94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds,
72.degree. C. for 1 minute, followed by extension at 72.degree. C.
for 10 minutes. Five to ten microliters of the nested reaction were
separated on 2% agarose gel (Pronarose, Hispanagar, Burgos, Spain).
Each assay included titrations of a murine erythroid leukemia cell
line C88-C1, containing a single provirus integration of IG-CFT
(21) and/or a titration of a pool of G418 selected MEL cells
infected with IG-CFT*. For practical reasons, we developed an
alternative PCR method to detect the neo-cassette in the
recombinant AAV-vectors IG-CFT, IG-CFT* and IG-.DELTA.Mo+NEO. The
sequences of the primers were as follows: NEO-1S:
5'-TAGCGTTGGCTACCCGTGAT- -3' (SEQ. ID. NO. 5), and NEO-4AS:
5'-TGCCGTCATAGCGCGGGTT-3' (SEQ. ID. NO. 6). Reaction mixtures were
prepared as described above and the reaction temperature was
95.degree. C. for 3 minutes followed by 30 cycles of 95.degree. C.
for 30 seconds, 65.degree. C. for 30 seconds and 72.degree. C. for
1 minute. The completion of the 30 cycles was followed by an
extension of 5 minutes at 72.degree. C. Five to ten microliter of
the PCR-reaction was run on a 2% agarose gel, blotted and
hybridized to a 157 bp specific probe isolated from a BstBI-SmaI
digest of IG-CFT.
[0084] The .beta.-globin specific PCR was carried out in
essentially the same way as the first reaction of the
neo.sup.R-specific PCR. But instead of the neo.sup.R-primers, the
primers listed below, specific for the 3' part of the HS-2 fragment
and .beta.-globin intron I, were added. The sequences of the
primers are:
2 HS-2-S3 5'-GGAATTATTCGGATCTATCGAT-3' (SEQ. ID. NO. 7) IVS-1A-A
5'-TCCTTAAACCTGTCTTGTAACC-3' (SEQ. ID. NO. 8)
[0085] The temperatures for the cycling were: 95.degree. C. for 3
minutes and then 30 cycles of 95.degree. C. for 30 seconds,
55.degree. C. for 30 seconds, 72.degree. C. for 30 seconds.
Following the 30 cycles, an extension at 72.degree. C. for 5
minutes was performed. Samples were run on 2% agarose gels, which
were blotted and hybridized to a NcoI-ClaI .beta.-globin promoter
specific probe using standard techniques.
[0086] Hemopoietic Data of the Transplantation of Rhesus Monkeys
with Recombinant AAV-transduced BMC
[0087] The survival and the selection of the purification and
transduction procedure of CD34.sup.+ rhesus monkey bone marrow
cells was controlled by determining the number of CFU-C present at
different stages in the procedure. The CD34 selection for Rh9128
and Rh9170 resulted in a 13-19 fold enrichment of CFU-C resp. For
A94 and BB94, the enrichment for CFU-C was 37-92 fold, respectively
(Table 4). The number of CFU-C did not vary by more than a factor
of 2 during culture or upon transduction, with the exception of
monkey BB94, where the decrease in the number of CFU-C was
considerable upon culture and infection with IG-CFT. This was due
to a direct toxicity of the CsCl-purified IG-CFT batch, as
determined by a titration of the batch on human cord blood post
ficoll bone marrow, which resulted in a dilution factor dependent
toxicity on CFU-C (not shown). Since it is known that CsCl is a
very toxic substance, we determined the CsCl concentration in the
two CsCl purified recombinant AAV preparations. Both contained
considerable amounts of CsCl, enough to account for the observed
toxicity (Table 2). Due to the observed toxicity on CFU-C in this
experiment, the two grafts that Rh BB94 received were very
different in size. Whereas the cultured graft was still
considerable, the graft-size for the short transduction protocol
was very small (Table 4). However, since stem cells are not
measured in a CFU-C assay and are indeed more resistant to a large
variety of drugs and agents, it is possible that many of them
survived the high concentration of CsCl.
[0088] Detection of Recombinant AAV Transduced Peripheral Blood
Cells
[0089] To determine whether the engrafted cells had been transduced
by the recombinant AAV vectors, approximately 3 ml of blood was
collected each week from every monkey. Granulocytes and mononuclear
cells were purified, as described in (57), and the DNA was released
and analyzed by PCR for the presence of recombinant AAV-sequences.
Two different PCR reactions were performed. On the samples from all
four monkeys, PCR reactions specific for the neo.sup.R-gene were
performed. The neo.sup.R-gene is present in all the vectors, so
this PCR detects all recombinant AAV-genomes present in the cells.
On the samples from monkeys rh-A94 and rh-BB94, also a
.beta.-globin specific PCR was performed. This PCR utilizes the
size difference in the .beta.-globin promoter in vectors IG-CFT and
IG-CFT*. These vectors were used to transduce the P-PHSC via two
different protocols. The effect of the two different protocols can
thus be read out by the prevalence of one of the two vectors in the
peripheral blood cells of the monkeys.
[0090] The results of the neo-PCR are depicted in FIGS. 2 and 3.
All monkeys were negative for recombinant AAV before
transplantation and became positive for recombinant AAV after
transplantation. The presence of the vector varied from week to
week. Some samples were positive for the vector, others were
negative, indicating that the frequency of transduced cells
averaged around the detection limit of the PCR-reaction which was
determined to be at 1 copy in 10.sup.5 nucleated cells for the
neo-specific PCR. Monkey BB94 was positive in all samples
immediately after transplantation and regeneration of the
hemopoietic system, indicating a more efficient transduction of
early progenitors during the ex vivo handling of the cells.
[0091] In monkeys BB94 and 9128, vector containing cells could be
detected for at least more than one year after transplantation.
Bone marrow samples taken from these animals at 2 and 6 months
(9128) or 14 months (BB94) post transplantation also contained
vector transduced cells. In BB94, the vector was detected in PBMC,
granulocytes, bone marrow and purified populations of B- and
T-cells (FIG. 4). This result demonstrated the transduction of stem
cells that had repopulated both the myeloid lineage (granulocytes)
and the lymphoid lineage (T- and B-cells). The granulocytes, T
cells, and B cells were still PCR positive more than 15 months
post-transplantation, indicating the transduction of cells with
extensive self-renewal capacity. The transduction of primate cells
with (1) an extremely long-term in vivo stability after
transplantation, and (2) the capability of multiple-lineage
repopulation long after transplantation, provides strong evidence
for transduction of P-PHSC.
[0092] Rhesus monkey 9128 received treatments with taxotere, a
cytostatic drug, to ablate the mature cells in the circulation,
inducing periodic regrowth from immature hemopoietic cells residing
in the bone marrow. Recombinant AAV transduced cells were detected
in circulating cells after a series of treatments with taxotere
over a period of 14 months post transplantation. The persistence of
transduced cells in peripheral blood cells and the resistance to
taxotere treatment provides convincing evidence of the transduction
of P-PHSC.
[0093] Determination of Most Efficient Transduction Protocol
[0094] The experiment with monkeys BB94 and A94 was designed to
quantify the success of two different transduction protocols. For
each monkey, the transplant was split in two equal fractions and
each fraction was transduced in a different way. To be able to
discriminate which protocol resulted in a better transduction, we
used a different vector for each transduction. We compared the
efficiency of transduction of cultured P-PHSC versus that of
non-cultured P-PHSC. For the transduction of P-PHSC from monkey
BB94, we used the purified virus IG-GFT for the non-cultured P-PHSC
and the purified virus IG-CFT* for the cultured P-PHSC. To exclude
a possible role of quality differences between the virus batches,
we switched the two virus batches for the transduction protocols
for monkey A94: we used IG-GFT for its cultured P-PHSC and IG-GFT*
for its non-cultured P-PHSC. Following transplantation and
repopulation of the hemopoietic system of the monkeys, we performed
the .beta.-globin specific PCR to determine which transduction
procedure resulted in the highest frequency of gene modified
circulating cells. For both monkeys, we were able to detect only
the virus used to transduce the cultured P-PHSC, i.e., IG-GFT* for
monkey BB94 and IG-GFT for monkey A94 (FIG. 5). Thus, in vitro
stimulation of P-PHSC results in a more efficient transduction with
recombinant AAV vectors. This result was not expected. It is
generally accepted that culture of P-PHSC promotes progressive loss
of the grafting potential of the P-PHSC, presumably due to
differentiation. Hence, if both procedures resulted in similar
P-PHSC transduction efficiencies, we would expect the progeny of
the non-cultured P-PHSC to prevail among the circulating blood
cells due to grafting advantages. Since we observed the opposite,
the stable transduction efficiency of the cultured P-PHSC must be
significantly higher than that of the non-cultured P-PHSC. It is
known that AAV-vectors integrate with higher efficiency in cycling
cells than in non-cycling cells (38). However, in non-cycling cells
the vector remains in the nucleus and retains its ability to
integrate when the cell is triggered into cycle (60). Once
transplanted, the P-PHSC start to divide and repopulate the ablated
hemopoietic system. Considering the enormous amount of cells that
need to be produced in a short time, it is presumed that the P-PHSC
start to divide within a couple of days once inside the body.
Therefore, a difference in transducibility of cultured versus
non-cultured cells is not expected when only replication of the
target cells is the enhancing factor. We thus infer that culture
and exposure to hemopoietic growth factors, such as IL-3, could in
other ways potentiate the transduction with recombinant AAV. One
possible explanation is the up-regulation or activation of
receptors for the virus on the surface of the P-PHSC. Another is
the induction of proteins inside the P-PHSC that enhance, for
instance, nuclear transport and/or other rate limiting steps for
stable transduction.
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3 TABLE 1 Amphotropic AAV Retrovirus Vector design Maximum insert
size 4.5 kb 8 kb Intron compatible Yes Poor Vector transcription in
packaging Not required Should be high cell Hemopoletic host range
Murine in vitro CFU Yes.sup.a Yes.sup.b Murine PHSC Not yet
reported Yes.sup.c Human in vitro CD34.sup.+ CFU Yes.sup.d
Yes.sup.e Human in vivo longlived Not yet reported Yes.sup.f
progenitors Provirus integrity Point mutations per viral genome*
0.005 1 Recombination frequency Insert-dependent Insert-dependent
Virus production Crude titers 10.sup.5 g 10.sup.7 h Concentrated
titers 10.sup.10 i 10.sup.8 j Helper free stocks Yes Yes Properties
of adeno-associated virus and amphotropic retrovirus vectors.
*Calculated number per replication cycle. AAV is replicated via
cellular DNA-polymerases which have proof reading activity. The
error frequency of these polymerases is 10.sup.-6 implying 1 point
mutation per 200 recombinant AAV genomes. Retroviruses are
replicated via RNA-polymerase II and reverse transcriptase (RT).
The known error frequency of RT is 10.sup.-4. Not much is known
about the mutation rate of RNA-polymerase II. Based on the error
frequency of RT one can expect one point mutation per retroviral
genome of 10 kb. .sup.a(Srivastava 1993); .sup.b(Joyner, 1983);
.sup.c(Einerhand 1992 #109); .sup.d(Chatteryee, 1992);
.sup.e(Nolta, 1992); .sup.f(Brenner, 1993); g (Walsh, 1992); h
(Miller, 1992); i (Flotte, 1993); j (Kotani, 1994; Lynch,
1991).
[0155]
4TABLE 2 Infectious Transducing wtAAV Adenovirus rAAV Particles
Particles titer ts149 C.sub.SCl vector Purification (IP/ml) (TP/ml)
(IP/ml) pfu/ml (mg/ml) IG-CFT Crude 2 .times. 10.sup.6 10.sup.4 4.5
.times. 10.sup.4 <10.sup.4 N.D. IG-.DELTA.Mo-Neo Crude 2 .times.
10.sup.7 10.sup.3 <10.sup.3 N.D. N.D. IG-CFT CsCl 10.sup.9 3.3
.times. 10.sup.5 10.sup.9 <10.sup.4 64 IG-CFT* CsCl 3 .times.
10.sup.8 3.3 .times. 10.sup.4 3 .times. 10.sup.9 <10.sup.4
44
[0156]
5TABLE 3 Rhesus Virus Time in no. of no. of monkey rAAV-vector
stock culture CD34.sup.+ IP TP IP/Cell TP/Cell 9170
IG-.sub..DELTA.Mo-Neo Crude 4 5 .times. 10.sup.6 10.sup.7 500 20
10.sup.-3 IG-CFT Crude 4 5 .times. 10.sup.6 10.sup.6 500 2
10.sup.-2 IG-.sub..DELTA.Mo-Neo 9128 IG-CFT Crude 4 9 .times.
10.sup.5 10.sup.7 500 20 10.sup.-3 Crude 4 9 .times. 10.sup.5
10.sup.6 500 2 10.sup.-2 BB94 IG-CFT* CsCl 4 4 .times. 10.sup.6 2
.times. 10.sup.7 2 .times. 10.sup.3 5 5 .times. 10.sup.-4 IG-CFT
CsCl 0 2 .times. 10.sup.6 1 .times. 10.sup.8 3.3 .times. 10.sup.4
50 2 .times. 10.sup.-2 A94 IG-CFT CsCl 4 6 .times. 10.sup.5 1.3
.times. 10.sup.6 430 2 4 .times. 10.sup.-4 IG-CFT* CsCl 0 2 .times.
10.sup.5 1.5 .times. 10.sup.6 160 7.5 8 .times. 10.sup.-4
[0157]
6TABLE 4 Time in CD34.sup.+ CFU-C Graft-size Reticulocyte Rhesus
Virus culture Cells per 10.sup.5 in CFU-C regeneration monkey
rAAV-vector stock (days) (.times. 10.sup.5) Cells (.times.
10.sup.3) date 9170 -- -- 0 100 1520 IG-.DELTA.Mo-Neo Crude 4 50
1480 74 IG-CFT Crude 4 50 900 45 22 9128 -- -- 0 18 940 16
IG-.DELTA.Mo-Neo Crude 4 9 1860 12 24 IG-CFT Crude 4 9 1400 BB94 --
-- 0 40 12000 IG-CFT* CsCl 4 40 2000 75 -- -- 0 20 16000 IG-CFT
CsCl 0 20 80 1.5 22 A94 -- -- 0 6 12 IG-CFT CsCl 4 6 23 130 -- -- 0
2 21 IG-CFT* CsCl 0 2 17 34 25
* * * * *