Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-04
2001-11-06
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S440000
Reexamination Certificate
active
06312957
ABSTRACT:
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 [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 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 [1,2,3]. Although long-term in vitro culture assays, such as, e.g., 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-PUSC 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 [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].
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.
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 virus adeno-associated 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 adenavirus 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].
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° 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].
Several characteristics distinguish AAV-vectors from the classical retroviral vectors (see also 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 &bgr;-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 introris during the RNA-stage of their life-cycle [23].
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 wtAAV [20]. Site-speciftc 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 inTmunogenicity problem associated with viral protein expression in transduced target cells. One drawback of AAV-ve
Einerhand Markus Peter Wilhelmus
Valerio Domenico
Davis Katharine F
IntroGene B.V.
Schwartzman Robert A.
TraskBritt PC
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