Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]

Abstract

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.

Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adeno-associated virus/human papillomavirus type 16 E7 antigen gene transduction into dendritic cells

“…AAV has been shown in various studies to be an effective gene delivery vector for both immortalized tissue culture cells as well as primary hematopoietic cells [15][16][17][18][19][20][21][22][23][24][25][26]. We recently showed that it is possible to successfully transduce, with chromosomal integration, the granulocyte macrophage-colony stimulating factor (GM-CSF) gene into monocytes (Mo) and DC via AAV [15].…”

Recombinant Adeno‐Associated Virus‐Based Vectors Provide Short‐Term Rather Than Long‐Term Transduction of Primitive Hematopoietic Stem Cells

“…AAV vectors have been proposed as an alternative to retroviral vectors for achieving efficient transduction of non-dividing cells [12]. However, the efficiency of integration of such vectors in hematopoietic cells is low despite the prolonged episomal persistence of rAAV [13]. Our data also suggest a transient integration or prolonged episomal expression of the transgene.…”

Gene transfer into hematopoietic progenitor and stem cells: Progress and problems

“…There is some evidence that AAV infection and even genomic integration may be independent of active cell cycling [67]. Wild-type AAV may preferentially integrate into a specific site on chromosome 19 and thus might have a lower chance of random insertional mutagenesis, although there is no evidence that recombinant vectors share this property [68,69). The host range of AAV is broad (human, monkey, mouse, etc.…”

“…Even drug resistance does not prove integration. Another group reported that 60-70% of human or rhesus CD34' cells exposed to rAAV carrying the P-galactosidase gene expressed the gene product in the nucleus [69]. Colonies grown from these cells were picked and estimated to have viral-associated DNA sequences at a copy number of one to two per cell by semi-quantitative PCR, implying but not proving integration.…”

“…Integration of wild-type AAV into the genome of progenitors was documented using inverted PCR analysis of individual colonies and interestingly only four of 27 showed site-specific integration into chromosome 19. Hence, site-specific integration may be a rare event even with wild-type virus, especially at high titer and under the artificial transduction conditions of the experiment [69].…”