AU7211201A - Genetic modification of primate hemopoietic repopulating stem cells - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 DIVISIONAL APPLICATION o .o• Name of Applicant: Actual Inventor(s): Address for Service: Invention Title: Introgene B.V.

Markus Peter Wilhelmus EINERHAND and Domenico VALERIO DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia "Genetic modification of primate hemopoietic repopulating stem cells" Details of Parent Application No: 50702/98 The following statement is a full description of this invention, including the best method of performing it known to me/us: Q:\Oper\Vpa\Septembcr\2001\2454864 divisional Introgene .255.doc 14/9/01 1A Title: Genetic modification of primate hemopoietic repopulating stem cells FIELD OF THE INVENTION 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 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 Though retroviral vector technology is still improving, progress in increasing the transduction of P-PHSC is slow. This is due to the fact that 0* a solution is not straightforward and that the P-PHSC cannot S 15 be identified by a rapid in vitro culture method Though culture of hemopoie-tic 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 20 [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 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 approx. up to 0.01-0.1% of the P-PHSC 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] A number of methods have been devised to improve the P-PHSC transduction by retroviral vectors such as 20 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.

In contrast to P-PHSC, murine PHSC are very easily ee 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 virus adenoassociated virus (AAV) 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 adendvirus 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 20 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 25 chromosome [14,15].

In an AAV-vector, the entire protein-coding domain 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 (Ihr, 560C). 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 Several characteristics distinguish AAV-vectors from the classical retroviral vectors (see also table 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 21]. Some genes, most notably the human p-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 lifecycle [23].

In human target cells, wild-type AAV integrates preferentially into a discrete region (19ql3.3-qter) of chromosome 19 [24,25,26] This activity might correlate with 20 rep-gene expression in the target cell, since it was found that the large rep-proteins bind to. the human integration site in vitro AAV-vectors do integrate with high efficiency into the host chromosomal DNA, however, thus far they do not share the integration site specificity of wtAAV 25 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 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 wtAAV which 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; wildtype 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 wtAAV 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 Such proviruses -cannot be rescued and thus provide an additional safety level of these vectors.

The first AAV-vectors were made by replacing part of the AAV-coding region with either the Chloramphenicol Acetyltransferase (CAT) or the neo R gene [16,17]. All of these 20 vectors retained either a functional rep- or a functional cap-coding region. Recombinant virus was generated by cotransfection with a plasmid containing a complete AAVgenome. The recombinant AAV-CAT virus conferred Chloramphenicol Acetyltransferase activity to 293 cells [16] 25 whereas the recombinant neoR virus conferred G418-resistance to Human-Detroit 6 cells, KB-cells and mouse L-cells [17] 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 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 neoR gene or the CAT gene were transferred and expressed efficiently in airway epithelial cells 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 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 In vivo, the vector DNA could be detected in 50% of the nuclei at 3 months postadministration. Although the prevalence of the vector decreased after this time point still of the nuclei were positive at the six months time point The presence of the vector correlated well with expression of RNA and recombinant protein which where still detectable at the six months follow up AAV-vector mediated gene transfer into murine hemopoietic cells was demonstrated by the conference of G418 resistance to murine in vitro colony forming units (CFU) following infection with a recombinant AAV-vector carrying 20 the neoR-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 25 Efficient gene delivery (up to 80%) into human hemopoietic progenitors and human CD34 cells with AAV-neoR vectors has also been reported [34,35,36,37]. These studies demonstrated that rAAV 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 rAAV 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 adeno-associated virus 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 rAAV in CD34+ cells and in vitro growing colonies (CFU-C) was demonstrated in 1996 by Fischer-Adams et al. 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 rAAV to P-PHSC, the only relevant hemopoietic cell type for long term persistence of transduced cells in vivo.

We are developing rAAV gene transfer into P-PHSC for the treatment of P-thalassemia and Sickle cell anemia. Both diseases severely affect the function of erythrocytes in these patients. p-thalassemic erythrocytes contain insufficient p-globin chains whereas mutant P-globin chains are made in sickle cell anemia (for review see Both diseases severely affect erythrocyte function which can be alleviated by persistent y-globin gene expression in the adult patient in which case fetal hemoglobin is formed Both inherited diseases are recessive in nature which 20 indicates that one functional intact copy of the adult pglobin gene is sufficient to ameliorate the phenotype.

Globin abnormalities were discarded as targets for gene S therapy attempts in the early days of gene therapy research.

This was largely due to the extremely complicated expression 25 patterns of globin-like genes Globin-synthesis is highly regulated during development and confined to cells of the erythroid lineage. Furthermore, the expression of a- and -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 p-globin gene with its promoter and local enhancer elements can direct erythroid specific globin RNA expression However, typically, the levels are less than 1% of the endogenous globin RNA.

Recently, sequences 50-60 kb upstream of the p-globin gene were discovered that direct the high level, tissue specific, copy number dependent and position independent expression of the p-globin gene This region, designated the Locus Control Region (LCR), is characterized by four strong erythroid-specific DNaseI hypersensitive sites (HS1- 4) 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 y-globin gene and the core fragment of HS2 together with the neoR gene into an AAV-vector Infected and G418 selected pools and clones of K562 cells produced the marked y-globin RNA to 50-85% compared to the normal level expressed by one endogenous y-globin gene [20,45]. A drawback of this vector is that the y-globin gene and promoter used in these studies are specific for expression in fetal erythroid tissue and thus not ideal for use as a therapeutic agent in adult humans. Incorporation of p-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 The 20 present invention describes the use of this and similar vectors in the transduction of P-PHSC.

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 wtAAV.

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 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 fragment thereof. Normally and preferably, but not necessarily, said DNA sequence will be a non-AAV DNA sequence, in particular a therapeutic DNA sequence.

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 a globin gene from the human p-globin gene cluster, a DNA sequence encoding an RNA or protein with anti-viral activity, the al-antitrypsin gene and the human multidrug resistance gene I (MDRI).

In a particularly preferred embodiment, the DNA S. sequence comprises the human p-globin gene inclusive of at 20 least one of its introns or functional analogs thereof, under transcriptional control of a functional part of the P-globin promoter or functional analogs thereof, and being operably linked to erythroid-specific DNaseI hypersensitive sites from its Locus Control Region (LCR), more particularly 25 the P-LCR elements HS4, HS3 and HS2 or functional analogs thereof.

The DNA sequence may also comprise a selectable marker gene useful in hemopoietic stem cells, such as a neoR gene, under transcriptional control of a herpes simplex virus (HSV) thymidine kinase (tk) promoter or functional analogs thereof or a AMo+PyFl01 Long Terminal Repeat (LTR) promoter.

The P-PHSC may be obtained from primate bone marrow, cord blood or peripheral blood, and preferably from a human.

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 The present invention is based on the discovery that adeno-associated virus-derived vectors efficiently transduce primate pluripotent hemopoietic stem cells. Adeno-associated virus 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. In addition, it is surprising that the vector integrates with high efficiency into P-PHSC even though most of the P-PHSC are not actively dividing at the time of infection. This is surprising since it has been established that rAAV integration in dividing cells occurs 200 times more efficiently in dividing as opposed to nondividing cells Also, it was reported that primary cells are much less efficiently transduced by rAAV than immortalized cell lines In addition it was reported that orf 6 from adenovirus E4-region stimulates transduction by recombinant AAV In a gene therapy setting it is undesirable to have functionally active adenovirus present 20 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, New Mexico Feb. 4-10, 1996, Prof. A. Nienhuis presented a paper stating that they transduced rhesus monkey CD34+ cells and subsequently 25 autologously transplanted the infected cells [49 Analysis of the peripheral blood cells circulating in blood with a polymerase chain reaction specific for the rAAV revealed that cells carrying the rAAV were only detected up until 7 days post transplantation i.e. P-PHSC were not transduced by rAAV in their experiment. Nonetheless, the present invention demonstrates that an adeno-associated virus-derived vector may be used to deliver exogenous DNA efficiently to cells of the hemopoietic system with long term repopulating ability.

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 primatederived 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. For this reason protocols to transduce hemopoietic progenitor cells with rAAV 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.

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 be packaged into a complex 20 before use. As used herein, the term "complex" is defined as a combination of two or more components physically linked to eachother through hydrophobic, hydrophilic or electrostatic interactions or covalent bonds, whereby one component of the complex at least is a recombinant AAV molecule. Other 25 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, the form of an intact virion or particle where the recombinant AAV vector is packaged inside an AAV capsid or functional analogs thereof.

As used herein, the term "functional analogs" refers to the same activity in kind, but not in amount or degree, i.e.

not quantitatively.

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 p-globin gene operably linked to cisacting sequences for erythroid specific physiological expression, the human lysosomal glucocerebrosidase gene .I the al-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 .i obtained from AAV serotypes 1, 2, 3, 4 or 5. Alternatively, 20 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. Packaging of rAAV into AAV-virions can be achieved using a variety of different methods. All methods are based on S 25 bringing the necessary proteins and rAAV-containing DNA in an environment that supports the replication and packaging of rAAV. One method relies on the transfection of adenovirus infected human cells with a plasmid carrying the rAAV-DNA together with a plasmid containing expression cassettes for the AAV-genes rep and cap. Upon continued culture of the manipulated cells, rAAV 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 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 [15-19]. 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.

The rep-gene can be derived from AAV serotypes 1-5, or functional analogues thereof either obtained through non- 20 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].

The cap-gene can be derived from AAV serotypes 1-5, or functional analogues thereof obtained through non-essential 25 mutations in the cap-genes. Alternatively, the cap-gene sequences-can be altered through the replacement or addition of sequences rendering the produced virion new or altered target cell specificities.

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.

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 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.

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 vive 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 20 suitable methods have been developed to target the recombinant AAV vector in vivo to P-PHSC.

S

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS Table 1 Key properties of Adeno-4ssociated virus 25 vectors and amphotropic retrovirus vectors.

Table 2 Characterization of recombinant

AAV

preparations useful for the transduction of primate

PHSC.

Table 3 Transduction of primate PHSC: culture and infection conditions.

IP Infectious Particles (titrated in RCA); TP Transducing Particles (titrated on MEL cells).

Table 4 Transduction of primate PHSC: Hemopoietic data.

Fig. 1A Recombinant AAV-vectors useful for the transduction of primate

PHSC.

ITR Adeno-associated virus inverted terminal repeat.

LCR= Core sequences from hypersensitive sites 4, 3 and 2 from the p-globin locus control region.

-103 human p-globin gene promoter fragment extending -103 upstream of the transcription start site.

-265 human p-globin gene promoter fragment extending -265 upstream of the transcription start site.

p-globin human P-globin gene with modified intron 2 (see text and 21).

Tkprom Herpes Simplex Virus Thymidine kinase gene promoter (approx. 500 bp NarI-BglII fragment).

NEO BglII-SmaI fragment from E. coli Tn5 transposon.

pA Polyadenylation signal from Herpes Simplex Virus Thymidine Kinase gene (approx. 500 bp SmaI-NarI fragment).

p*-globin human p-globin gene with in the 5' untranslated region three point mutations that generate two restriction enzyme sites (see fig. 1B) AMo+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] Fig. 1B Nucleotide sequence of the 5' untranslated region (UTR) of the normal and the marked human p-globin gene.

Fig. 2 Detection of recombinant AAV in rhesus monkey S 25 peripheral blood cells. Blood cells were collected as described in the text. Peripheral blood mononuclear cells (WBC) were separated from the granulocytes (Gran) and a neospecific 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 rAAV-vector could be detected in a background of 105 negative cells. indicates the presence of a neo-specific band and the absence of a neo-specific band in the agarose gel.

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 figure 2 are included in figure 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 figure 3b indicate the time-points at which docetaxel (Taxotere) was administered.

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).

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 pb. longer than the fragment specific for

IG-CFT.

EXAMPLE 1 Ligation of recombinant AAV vectors containing the human p-globin gene and/or the neoR gene S 25 In order to determine whether recombinant AAV could transduce P-PHSC it was necessary to generate appropriate vectors. We generated three different recombinant AAVvectors, which are schematically represented in figure 1A.

The ligation of the vector IG-CFT containing a human P-globin gene together with sequences from the p-globin locus control region and the neoR-gene is described in IG-CFT* differs from IG-CFT in the size of the human p-globin promoter and in the presence of three point mutations in the untranslated region (UTR) of the human P-globin gene. In IG-CFT* the promoter driving p-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 UTR of the human p-globin gene created two new restriction sites, one for XbaI and one for HindIII, see also figure lB.

IG-AMoNeo (depicted in fig. 1A) contains the rAAVbackbone (XbaI-fragment) from pSub201 the NheI-Smal promoter-fragment from the AMo+PyF101 LTR the BglII- SmaI fragment from the Tn5-derived neoR-gene followed by the SmaI-NarI poly-adenylation signal from Herpes Simplex Virus (HSV) Thymidine Kinase (TK) gene The elements were linked together using the polylinker from pBluescript SK+ (Stratagene).

Example 2 Production of recombinant AAV from IG-CFT, IG-CFT* and IG-AMoNeo The 293 cell line which is a human embryonic S kidney cell line transformed with Ad5 DNA, the A549 cell line, which is a human bronchial carcinoma cell line, and the C88 cell line which is a murine erythroleukemia (MEL) cell line, were maintained in DMEM (GIBCO-BRL) containing 10% Fetal Calf Serum (FCS), 100 pg/ml streptomycin and 100 U/ml penicillin. Recombinant AAV was produced by transfecting a rAAV packaging plasmid and a vector plasmid into approx. 90% confluent permissive 293 cells. The cells 25 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 wtAAV2 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 upon expression from the packaging plasmid replicates 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 0 C water bath. Cell debri was subsequently pelleted (10 min, 200g) and the supernatant was incubated at 56 0 C for 1 hour to inactivate residual adenovirus. Concentrated high titer recombinant AAV stocks were prepared by harvesting the cells in there own culture medium, and washing in PBS (max. 107 cells/ml). The virus was released from the cells by 3 freeze thaw cycles and/or sonication pulses of 1 second on ice to prevent warming.

Cell debri was spun down and the supernatant was made a density of 1.4 by adding solid CsCl. After o/n centrifugation (50.000 20 0 C, using a vti TI65.1 rotor in a Beckman ultracentrifuge) fractions were collected and rAAV was determined. Fractions containing rAAV 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 see**: Characterization of rAAV preparations To determine the effect of the different methods of 25 virus preparation and the different processing steps on the quality of the various rAAV-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 S"center assay (RCA) described below, 2) the capacity to stably transduce cells and express the neoR-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 wtAAV, 4) the amount of replication proficient adenovirus in each preparation, and 5) the concentration of CsCl in the rAAV preparations that were purified using CsCl gradients (See Table 2).

Replication center assay The replication center assay (RCA) takes advantage of the fact that in a lytic infection of AAV up to 10 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 ts149 20) and wtAAV-2 For a titration of the wild type AAV the cells were infected with dilutions of recombinant virus stock and adenovirus ts149 20). The cells were subsequently incubated at 39 0

C.

The next day after 24 hours the medium was replaced by icecold PBS containing 5 mM EDTA. After 5 to 20 min. on ice a single cell suspension was made by rigorous pipetting. The cells were diluted in 5 ml PBS and sucked onto hybond N+ filter circles (pore size 0.22 RM) of 3.6 cm diameter.

Filters were incubated for 5 min in denaturation solution (0.4 M NaOH; 0.6 M NaC1) and 5 min in renaturation buffer (1.5 M NaC1; 1 M Tris-HCl, pH Filters were washed and stored in 5xSSPE 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.

MEL-cell transduction x 105 MEL cells were seeded in 2 ml culture medium per well (24 wells plate, Falcon) and the appropriate dilution of rAAV virus was added. The cells were collected the next day and reseeded in 30 ml culture medium in a 75 cm 2 flask (Falcon). After three days the medium was replaced by selection medium by spinning down the cells (200g, 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 rAAV capable of stably transducing MEL cells. Specific virus dilutions were scored negative when after fourteen days confluency had not been reached.

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.

A summary of the characterization is given in Table 2.

The infectious particle (IP) concentration, i.e. the capacity -to deliver rAAV-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 a IP to TP ratio of •20 104, the 248 crude batch had a much lower ratio of 200.

Example 4 Transduction and autologous transplantation of rhesus monkey bone marrow S 25 Animal care and transplantation 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 mg/kg/day) during 5 days, followed by daily oral administration of ciprofloxacin (6.5 mg/kg/day), polymixin B mg/kg/day) and nystatin (40 kU/monkey/day). A94 and BB94 received one administration of ivermectine 200 pg/kg antiworm treatment approximately two weeks prior to transplantation. The monkeys were kept under barrier nursing and antimicrobial treatment until leukocyte counts exceeded a value of Ixl0 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 40x10 9 /liter, subcutaneous fluid upon indication and red blood cell transfusions when hematocrit levels dropped below 0.2 1/1. 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®) treatment was given to monkeys rh9128 and rh9170 at indicated times (fig. 3) at a dose of 50 mg/m 2 In monkey rh9128 around 14 months post transplantation 4 docetaxel doses were given of 10 mg/m 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.

Bone marrow processing and transduction 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 selection was performed using a smallscale CEPRATE LC column (CellPro, Bothell, WA). From 5x10 4 to 50x10 4 cells were incubated at 4 0 C for 30 min 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+ cells was calculated as the ratio of CD34+ cells to total number of cells and multiplied by 100. For rhesus monkeys 9128 and 9170 the enriched CD34+ cells were immediately processed for transduction. Forrhesus monkeys A94 and BB94 the enriched CD34+ cells were 20 split into two equal fractions and stored in liquid nitrogen.

Transduction of CD34+ cells was done as described below.

A summary of the experimental conditions is given in table 3.

25 Rhesus monkey 9128 and 9170: Four days prior to transplantation the CD34+ enriched cells were split in two equal fractions and cultured at a density of 106 cells per ml in low density BMC culture medium supplemented with recombinant rhesus monkey interleukin-3 (RhIL-3; Burger et al., 1990) as 30 described in On day 2 and day 3 one fraction of cultured CD34+ cells was exposed to the crude rAAV preparation of IG-CFT and the other fraction was exposed to a crude rAAV-preparation of IG-AMoNeo by adding an equal volume of virus preparation to the medium of the cultured CD34+ cells. After three to five hours the cells were collected by centrifugation (7 min, 200g) 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, 200g) and resuspended in an equal volume of 0.9% NaCl and separately transplanted into autologous rhesus monkeys by IV injection.

Rhesus monkey A94 and BB94: Four days prior to transplantation one fraction of the frozen CD34+ 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 106 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, 200g) and resuspended in 10 to 200 pi of IMDM+10%FCS and RhIL-3 and subsequently exposed to a purified rAAV 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 20 four the cells were collected by centrifugation and suspended in 0.9% NaC1. Also on day four the other fraction of CD34+ cells was thawed and washed with Hanks Balanced Salt *solution. Live cells were collected by Ficoll-Hypaque (Sigma) centrifugation, resuspended in 10 to 200 pl of "25 IMDM+10%FCS and RhIL-3 and subsequently exposed to a purified rAAV-preparation of IG-CFT (Monkey BB94) or IG-CFT* (Monkey A9.4). After two hours the cells were collected by centrifugation and suspended in NaCl. After collection in NaCl the cells were separately transplanted into autologous irradiated rhesus monkeys by IV injection.

Parameter evaluation 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 24 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.

FITC

labeled CD2 (clone S 5.2; Becton-Dickinson, California) or (clone L27; Becton-Dickinson, California) antibodies were incubated with PBMC according to the manufacturers protocols. Labeled cells were separated using the MACS® column and anti-FITC beads (Miltenyi, Germany) according to the manufacturers protocol. Re-analyses of the sorted cells on FACS® (Becton-Dickinson, USA) showed that the sorted cells were more then 95% pure populations.

Colony-forming cell (CPFT-C) assay Rh9128 and Rh9170 hemopoietic cells were plated in duplicate at 5 x 10 3 /ml (CD34+ selected) or Ixl0 5 /ml (post- Ficoll) in 1 ml methylcellulose medium as described in [57] 20 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).

Polymerase chain reaction For cell lysis, pellets were incubated (107 cells/ml) in nonionic detergent lysis buffer NP40, 0.5% Tween mM Tris pH 8.3, 50 mM KC1, 0.01% gelatin, 2.5 mM MgCl 2 containing proteinase K (60 mg/ml) at 56 0 C for 1 hour.

Lysates were then heated at 95 0 C for 10 min to inactivate the proteinase K. Two different PCR detections were performed. One was a nested neoR-specific PCR and one was a p-globin specific PCR. The protocol for the neoR-specific

PCR

will be described first. The first amplification was performed on 10 i lysates in a total volume of 50 1i with 2 U of SuperTaq polymerase (HT Biotechnology, Cambridge, England) in a reaction mix (final concentration: 200 mM each of 2'-deoxyadenosine-5'-triphosphate, triphosphate, 2'-deoxyguanosine-5'-triphosphate, 2'- (Pharmacia, Roosendaal, The Netherlands), 0.2 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 pl of the first reaction in a total volume of 50 il with 2 U of SuperTaq polymerase (HT Biotechnology, Cambridge, England) in a reaction mix (final concentration: 200 mM each of 2'-deoxyphosphate (Pharmacia, Roosendaal, The Netherlands), 0.2 iM 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 neoR gene.

The primer sequences are: 5' neo-1: 5' -GGGGTACCGCCGCCGCCACCATGATTGAACAAGATGGATTGC-3' 5' neo-2: 5'-TTCTCCGGCCGCTTGGGTGG-3' 3' neo-1: 5'-GGCAGGAGCAAGGTGAGATG-3' 3' neo-2: 5'-CCATGATGGATACTTTCTCG-3' Amplification conditions were the same for the 25 first and the nested amplification and were performed in a TRIO thermocycler (Biometra, G6ttingen, Germany) temperature cycling apparatus. The conditions chosen were: 950C for minutes, then 30 cycles of 94 0 C for 30 seconds, 55 0 C for seconds, 720C for 1 minute, followed by extension at 720C 30 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 rAAV-vectors IG-CFT, IG-CFT* and IG-AMo+NEO. The sequences of the primers were as follows: NEO-1S: 5'-TAGCGTTGGCTACCCGTGAT-3', and NEO-4AS: 5'-TGCCGTCATAGCGCGGGTT-3'. Reaction mixtures were prepared as described above and the reaction temperature was 95 0 C for 3 minutes followed by 30 cycles of 95°C for seconds, 65 0 C for 30 seconds and 72 0 C for 1 minute. The completion of the 30 cycles was followed by an extension of minutes at 72 0 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.

The p-globin specific PCR was carried out in essentially the same way as the fiTst reaction of the neoR-specific PCR.

But instead of the neoR-primers, the primers listed below specific-for the 3' part of the HS-2 fragment and p-globin intron I were added. The sequences of the primers are: HS-2-S3 5'-GGAATTATTCGGATCTATCGAT-3' IVS-1A-A 5'-TCCTTAAACCTGTCTTGTAACC-3' The temperatures for the cycling were: 95 0 C for 3 minutes 20 and then 30 cycles of 95 0 C for 30 seconds, 55 0 C for seconds, 72 0 C for 30 seconds. Following the 30 cycles, an extension at 72 0 C for 5 minutes was performed. Samples were run on 2% agarose gels, which were blotted and hybridized to a NcoI-ClaI p-globin promoter specific probe using standard 25 techniques.

Hemopoietic r data of the transplantation of rhesu.s monkeys with rAAV-transduced

BMC

The survival and the selection of the purification and transduction procedure of CD34+ 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 resp. (table The number of CFU- C did not vary by more then 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 rAAV preparations.

Both contained considerable amounts of CsCl, enough to account for the observed toxicity (table 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 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 CsC1.

Detection of rAAV transduced peripheral blood cells 20 To determine whether the engrafted cells had been transduced by the recombinant AAV vectors, approx. 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 25 PCR for the presence of rAAV-sequences. Two different PCR reactions were performed. On the samples from all four monkeys, PCR reactions specific for the neoR-gene were performed. The neoR-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 p-globin specific PCR was performed. This PCR utilizes the size difference in the -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.

The results of the neo-PCR are depicted in figures 2 and 3. All monkeys were negative for rAAV before transplantation and became positive for rAAV 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 105 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.

In monkeys BB94 and 9128 vector containing cells could be detected for at least more then 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 S 20 marrow and purified populations of B- and T-cells (Fig. 4).

This result demonstrated the transduction of stem cells which had repopulated both the myeloid lineage (granulocytes) and the lymphoid lineage and B-cells).

The granulocytes, T cells, and B cells were still PCR 25 positive more than 15 months post-transplantation, indicating the transduction of cells with extensive selfrenewal capacity. The transduction of primate cells with (1) an extremely long-term in vivo stability after transplantation, and the capability of multiple-lineage 30 repopulation long after transplantation, provides strong evidence for transduction of P-PHSC.

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.

Determination of most efficient transduction protocol 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 20 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 P-globin specific PCR to determine which transduction procedure 25 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. 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 noncultured P-PHSC. It is known that AAV-vectors integrate with higher efficiency in cycling cells then in non-cycling cells However, in non-cycling cells the vector remains in the nucleus and retains its ability to integrate when the cell is triggered into cycle 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-ultured cells is not expected when only replication of the target cells is the enhancing factor. We 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 *e 20 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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Proc. Natl. Acad. Sci. USA 91: 8915-8919, 1994 39. Wintrobe MM, Lee GR, Boggs DR, Bithell TC, Foerster J, Athens JW, Lukens JN: Clinical hematology: 869-903, 1981 Rodgers GP, Dover GJ, Noguchi CT, Schlechter AN: Hematologic responses of patients with sickle cell disease to treatment with hydroxyurea. N. Engl. J. Med. 322: 1037-1045, 1990 41. Anderson WF: Prospects for human gene therapy. Science 226: 401-409, 1984 20 42. Evans T, Felsenfeld G, Reitman M: Control of globin gene transcription. Ann. Rev. Cell Biol. 6: 95-124, 1990 43. Grosveld F, Blom van Assendelft G, Greaves DR, Kollias G: Position-independent, high-level expression of the human P-globin gene in transgenic mice. Cell 51: 975-985, 1987 25 44. Talbot D, Collis P, Antoniou M, Vidal M, Grosveld F, Greaves DR: A dominant control region from the human p-globin locus conferring integration site independent gene expression. Nature 338: 352-355, 1989 45. Miller JL, Walsh CE, Ney PA, Samulski RJ, Nienhuis AW: Single-copy transduction and expression of human y-globin in K562 erythroleukemia cells using recombinant adenoassociated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer. Blood 82: 1900-1906, 1993 46. Einerhand MPW, Valerio D: Viral vector systems for bone marrow gene therapy, in Levitt, Mertelsmann (eds): Hematopoietic Stem Cells: Biology and therapeutic applications, New York, Marcel Dekker, inc, 1995, p 275-295 47. Halbert CL, Alexander IE, Wolgamot GM, Miller AD: Adenoassociated virus vectors transduce primary cells much less efficient than immortalized cells. J. Virol. 69: 1473-1479, 1995 48. Ferrari FK, Samulski T, Shenk T, Samulski RJ: Second strand synthesis is a rate limiting step for efficient transduction by recombinant adeno-associated virus vectors.

J. Virol. 70: 3227-3234, 1996 49. Nienhuis A: Gene Therapy for Hematopoietic Stem Cells in Genetic Disease and Cancer. Keystone Symposia on Molecular and Cellular Biology, Taos, New Mexico Feb. 4-10, 1996 Lefebvre RB, Riva S, Berns KI: Conformation takes precedence over sequence in adeno-associated virus DNA replication. Mol. Cell Biol. 4: 1416-1419, 1984 51. Van Beusechem VW, Kukler A, Einerhand MPW, Bakx TA, Van der Eb AJ, Van Bekkum DW, Valerio D: Expression of human adenosine deaminase in mice transplanted with hemopoietic 20 stem cells infected with amphotropic retroviruses. J. Exp.

Med. 172: 729-736, 1990 52. Valerio D, Einerhand MPW, Wamsley PM, Bakx TA, Li CL, Verma IM: Retrovirus-mediated gene transfer into embryonal carcinoma cells and hemopoietic stem cells: Expression from 25 a hybrid long terminal repeat. Gene 84: 419-427, 1989 53. Linney E, Davis B, Overhauser J, Chao E, Fan H: Nonfunction of a Moloney Murine Leukemia Virus regulatory sequence in F9 embryonal carcinoma cells. Nature 308: 470- 472, 1984 54. McKnight S: The Nuceotide sequence and transcript map of the herpes simplex thymidine kinase gene. Nucl. Acids Res.

8: 5949-5964, 1980 Graham FL, Smiley J, Russell WC, Naiva R: Characteristics of a human cell line transformed by DNA from adenovirus type 5. J. Gen. Virol. 36: 59-72, 1977 56. Deisseroth A, Hendrick D: Human p-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells. Cell 15: 55-63, 1978 57. Van Beusechem VW, Kukler A, Heidt PJ, Valerio D: Longterm expression of human adenosine deaminase in rhesus monkeys transplanted with retrovirus-infected bone-marrow cells. Proc. Natl. Acad. Sci. USA 89: 7640-7644, 1992 58. Thomson BJ, Weindler FW, Gray D, Schwaab V, Heilbronn: Human Herpesvirus 6 (HHV-6) is a helpervirus for adenoassociated virus type 2 (AAV-2) and the.AAV-2 rep gene homologue in HHV-6 can mediate AAV-2 DNA replication and regulate gene expression. Virol. 204: 304-411, 1994 59. Fischer-Adams G, Wong JR KK, Podsakoff G, Forman Sj, Chatterjee

S

Integration of Adeno-associated virus vectors in CD34+ human hemopoietic progenitor cells after transduction. Blood 88: 492-504, 1996 Podsakoff G, Wong JR KK, Chatterjee S: Efficient gene transfer into nondividing.cells by adeno-associated virusbased vectors. J Virol 68: 5656-5666, 1994 S 20 Throughout this specification, unless the context requires therwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or 25 integer or group of elements or integers.

25 The reference to any prior art in this specification is not, Sand should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

I 1 9 9 9 Table 1 Amphotropic Retrovirus vector design Maximum insert size 4.5 kb Intron compatible Yes Vector transcription in packaging cell Not required Hemopoietic host range Murine in vitro CFU Murine PHSC Human in vitro CD34+ CFU Human in vivo longlived progenitors Provirus integrity Point mutations per viral genome* Recombination frequency Yesa Not yet reported Yes d Not yet reported 0.005 Insert-dependent 8 kb Poor Should be high Yesb Yes c Yes e Yesf Insert-dependent Insert-dependent Virus production Crude titers 105 g Concentrated titers 1010 i 107 h Helper free stocks Yes 108 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 10-6 implying 1 point mutation per 200 recombinant AAV genomes. Retroviruses are replicated via RNApolymerase II and reverse transcriptase The known error frequency of RT is 10-4 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.

a[Srivastava, 1993 b[Joyner, 1983]; C[Einerhand 1992 #109]; d[Chatteryee 1992]; e[Nolta 1992]; f[Brenner, 1993]; 9[Walsh, 1992]; h[Miller 1992]; i[Flotte, 1993] 1jKotani, 1994; Lynch, 19911.

9* S S S 555 Table 2 rAAV vector Purification Infectious Particles (IP/ml) Transducing Particles (TP/ml) WtAAV titer (TD/ -1 Adenovirus ts149 CsCl (mq/ml) IG-CFT Crude IG-AMo-Neo Crude 2 x 106 2 x 107 109 3 X108 104 103 4.5 x 104 <103

IG-CFT

IG-CFT*

CsC1 CsCl <104

N.D.

<104 <104 3.3 x 105 3.3 X 104 109 3 x 109

N.D.

D.

64 44 Table 3 Rhesus rAAV-vector Virus Time in CD34+ no. of no. of IP TP monkey stock culture Cells IP TP Cell Cell 9170 IGAo-- 9128 BB94 A94

IG-CFT

IG-AMo-Neo

IG-CFT

IG-CFT*

IG-CFT

IG-CFT

IG-CFT*

LUUd q Crude 4 5x0 6 107 5 x 106 106 Crude Crude CsCl CsCl CsCl CsCl 9x105 107 9 x 105 106 4 x106 2 x 107 2x10 6 1x108 500 500 500 500 2 x 103 3.3 x 104 430 160 20 2 2 5 so 2 7.5 10-3 10-2 10-3 10-2 5 x 10-4 2 x 10-2 4 x 10-4 8 x 10-4 6 x 105 2 x105 1.3 x 106 1.5 x 106 9 9. 9 90 Oe* 9** 000* 9 9.

9 0 00 9 0 9 0 9 9 0 0 0 00 *0 0 0 0 9 9 0 9 0 *9* Table 4 Rhesus rAAV-vector Virus Time in CD34+ CFU-C Graft-size Reticulocyte monkey stock culture Cells per 10 5 in CFU-C regeneration (days) (X 105) Cells (X 103) date 9170 9128 BB 94 A94 IG-AMo-Neo I G -CFT IG-AMo-Neo

IG-CFT

IG-CFT*

IG-CFT

IG-CFT

IG-CFT*

Crude Crude Crude Crude CsCl CsCl CsCl CsCl 100 50 50 1520 1480 900 940 1860 1400 12000 2000 16000 80 130

Claims (24)

1. A process of genetic modification of pluripotent hemopoietic stem cells of primates (P-PHSC), comprising introducing a recombinant adeno-associated virus (AAV) vector into P-PHSC.

2. The process of claim 1, wherein the recombinant AAV is derived from human AAV.

3. The process of claim 1 or claim 2, wherein the recombinant AAV vector comprises a DNA sequence flanked by the inverted terminal repeats (ITR) of AAV or functional analogs or fragments thereof.

4. The process of claim 3, wherein said DNA sequence comprises regulatory sequences which are functional in hemo- poietic cells and, under the control of said regulatory sequences, a sequence coding for a protein or RNA with a 5 therapeutic property when introduced into hemopoietic cells.

5. The process of claim 3 or claim 4, wherein said DNA sequence comprises the coding sequence of a gene selected from the group consisting of the human lysosomal glucocerebrosidase gene a globin gene from the human P-globin gene cluster, a DNA sequence encoding an RNA or protein with anti-viral activity, the al-antitrypsin gene and the human multidrug resistance gene I (MDRI).

6. The process of any one of claims 3 to 5, wherein said DNA sequence comprises the human P-globin gene inclusive of at least one intron or a functional analog thereof.

7. The process of any one of claims 3 to 6, wherein said DNA sequence comprises the human p-globin gene operably linked to erythroid-specific DNaseI hypersensitive sites from its Locus Control Region (LCR) or a functional analog thereof.

8. The process of claim 7, wherein said erythroid- specific DNaseI hypersensitive sites from the p-globin LCR comprise the p-LCR elements HS4, HS3 and HS2, or functional analogs thereof.

9. The process of any one of claims 3 to 8, wherein said DNA sequence comprises the human p-globin gene under transcriptional control of a functional part of the p-globin promoter or a functional analog thereof.

The process of any one of claims 3 to 9, wherein said DNA sequence comprises a selectable marker gene useful in hemopoietic stem cells.

11. The process of claim 10, wherein said selectable marker gene is a neoR gene under transcriptional control of a herpes simplex virus (HSV) thymidine kinase (tk) promoter or a functional analog'thereof.

12. The process of claim 10, wherein said selectable marker gene is a neoR gene under transcriptional control of a AMo+PyFl01 Long Terminal Repeat (LTR) promoter or a functional analog thereof.

13. The process of any one of claims 1-12, wherein said recombinant AAV vector is part of a complex when 20 contacted with said P-PHSC.

14. The process of claim 13, wherein said recombinant AAV vector is associated with AAV capsid proteins.

15. The process of claim 13, wherein said recombinant AAV vector is packaged into an AAV capsid. 25

16. The process of claim 15, wherein said recombinant AAV vector is introduced into said P-PHSC by transduction with the recombinant AAV vector packaged in an AAV capsid or a functional analog thereof.

17. The process of any one of claims 1 to 16, wherein said P-PHSC are obtained from primate bone marrow, cord blood or peripheral blood.

.18. The process of any one of claims 1 to 17, wherein said P-PHSC are obtained from a human.

19. The process of any one of claims 1 to 18, wherein said P-PHSC are exposed in vitro to one or more proliferation stimulating compounds.

The process of claim 19, wherein said P-PHSC are exposed in vitro to interleukin 3 or a functional analog or a fragment thereof.a og o

21. A primate pluripotent hemopoietic stem cell (p PHSC) obtainable by a process according to any one of the aforegoing claims.

22. A P-PHSC genetically modified by a process according to any one of claims 1-20.

23. A cell derived from a P-PHSC according to claim 21- or claim 22.

24. The process according to any one of claims 1-20, or the stem cell according to claim 21, or the process according to claim 22, or the cell according to claim 23, substantially as herein before described with reference to the figures and/or examples. SDATED this 14th day of September 2001 Introgene B.V. DAVIES COLLISON CAVE Patent Attorneys for the applicant *S *0 S