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 h… Show more
“…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.…”
Recombinant adeno-associated virus encoding the monhematocrits reached 62 and 75% by week 10 (from prekey erythropoietin gene (rAAV-cm-Epo) was generated injection values of 38 and 40%, respectively) and remained and tested for its potential to confer long-term expression elevated throughout the study period (28 weeks). Circulatof the gene product following intramuscular injection. A ing Epo levels were also elevated throughout the study persingle intramuscular injection of 2 × 10 12 rAAV-cm-Epo pariod. Our data demonstrate the potential for long-term gene ticles into two baboons led to sustained high circulating expression in large animals by a single intramuscular injecEpo levels and a concomitant increase in hematocrit. The tion of a recombinant adeno-associated virus (rAAV) vector.
“…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.…”
Recombinant adeno-associated virus encoding the monhematocrits reached 62 and 75% by week 10 (from prekey erythropoietin gene (rAAV-cm-Epo) was generated injection values of 38 and 40%, respectively) and remained and tested for its potential to confer long-term expression elevated throughout the study period (28 weeks). Circulatof the gene product following intramuscular injection. A ing Epo levels were also elevated throughout the study persingle intramuscular injection of 2 × 10 12 rAAV-cm-Epo pariod. Our data demonstrate the potential for long-term gene ticles into two baboons led to sustained high circulating expression in large animals by a single intramuscular injecEpo levels and a concomitant increase in hematocrit. The tion of a recombinant adeno-associated virus (rAAV) vector.
“…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.…”
Section: The Induced and The Uninduced Cells Were Then Transferred mentioning
confidence: 99%
“…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.…”
Section: Introductionmentioning
confidence: 99%
“…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.…”
“…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.…”
We examined the ability of recombinant adeno-associated no detectable constitutive expression of luciferase and that virus (rAAV) to transfer regulated gene expression into T luciferase gene expression remained inducible for at least cell lines. An AAV-based vector containing the neomycin 180 days. Luciferase expression was activated by PMA resistance gene and expressing the firefly luciferase (luc) and ionomycin and by anti-CD3 antibodies and was gene under the regulatory control of the interleukin 2 proinhibited by cyclosporin A. Examination of G418-resistant moter (pAAV-luc) was generated and adenovirus-free clones showed that rAAV-luc had integrated into the host rAAV (rAAV-luc) was produced from this vector. Transfecchromosomes but that some of the clones lost some of tion of pAAV-luc into the human T cell line Jurkat resulted the transferred DNA or lost expression from the transferred in luciferase expression while infection of Jurkat T cells DNA. These results indicate that rAAV can transfer and with rAAV-luc resulted in significant luciferase expression integrate regulated gene expression into T cell lines but only after selection for neomycin-resistant cells. Long-term that the transferred genetic material may be lost or its growth of transduced Jurkat T cells showed that there was expression may be silenced over time.
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