Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood.

Abstract

StlmmlryRecombinant adeno-assodated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoa), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human fl-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34 + cells isolated from human umbilical cord blood. In donogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neo p" gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit--erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-spedfic DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected calls, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these calls before transplantation.

Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates

“…29,30 Briefly, human embryonic kidney 293 cells, grown to 60% confluence in a 15-cm dish, were co-transfected with 12.5 g of helper plasmid pKS rep/cap and 12.5 g of vector plasmid pCMVAAV-cm-Epo by the calcium phosphate co-precipitation method. The precipitate was left on the cells for 8 h and the medium was replaced with IMDM + 10% FBS containing adenovirus type 5 dl312 at a multiplicity of infection (MOI) of 2.…”

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38− subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

“…We observed a similar trend in K562 cells 17 where very high MOIs were required for stable integration of the rAAV transgene and these findings have been independently confirmed by others. 26 Some investigators however, report efficient and long-term transduction of human 9,14,28,36,55 and murine hematopoietic progenitors 20 using relatively low rAAV MOIs. Even allowing for variGene Therapy ation in vector design and reporter genes there are several notable differences in the way these studies were performed.…”

“…8 Early studies suggested that rAAV vectors could transduce primitive human hematopoietic cells. [9][10][11][12][13][14] However, these studies were performed with crude vector preparations composed of lysate derived from producer cells, which had been infected with adenovirus and co-transfected with the vector plasmid and a packaging plasmid encoding the AAV proteins. Such preparations contain wild-type AAV, which arises by virtue of non-homologous recombination between the vector and packaging plasmids.…”

“…11 Previous studies with rAAV in tissue culture cells utilized selection to recover drug-resistant clones, which were then shown to contain an integrated pro-viral genome. 14,20 Such studies are totally uninformative with regard to the efficiency of rAAV integration. More recent studies have unequivocally demonstrated that vector uptake, conversion of the single-stranded to a doublestranded genome, and episomal-mediated, rAAV gene expression can occur without genome integration.…”

Transfer of activation-dependent gene expression into T cell lines by recombinant adeno-associated virus

“…4 AAV infects and integrates into the chromosomes of a wide range of host cells. [5][6][7][8] Recombinant AAV (rAAV) is generated from AAVbased plasmids in which all of the genomic DNA, except for the ITR, has been replaced by the desired gene therapy construct. 5,9 Recombinant AAV can also integrate into the genome of a wide range of host cells including lymphoid cells, 5,[10][11][12] and does not replicate and cannot be rescued upon superinfection with adenovirus unless wild-type AAV is also present.…”