Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells.

Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. We have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. We report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy.     

Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells.

Recombinant retroviruses encoding human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans.     

Generation of mature hematopoietic cells from human pluripotent stem cells

A number of malignant and non-malignant hematological disorders are associated with the abnormal production of mature blood cells or primitive hematopoietic precursors. Their capacity for continuous self-renewal without loss of pluripotency and the ability to differentiate into adult cell types from all three primitive germ layers make human embryonic stem cells and induced pluripotent stem cells (hiPSCs) attractive complementary cell sources for large-scale production of transfusable mature blood cell components in cell replacement therapies. The generation of patient-specific hematopoietic stem/precursor cells from iPSCs by the regulated manipulation of various factors involved in reprograming to ensure complete pluripotency, and developing innovative differentiation strategies for generating unlimited supply of clinically safe, transplantable, HLA-matched cells from hiPSCs to outnumber the inadequate source of hematopoietic stem cells obtained from cord blood, bone marrow and peripheral blood, would have a major impact on the field of regenerative and personalized medicine leading to translation of these results from bench to bedside.     

Modeling human hematopoietic cell development from pluripotent stem cells

Understanding the steps and cues that allow hematopoietic cells to be generated during development holds great clinical as well as biological interest. Analysis of these events in mice has provided many important insights into the processes involved, but features that might be unique to humans remain challenging to elucidate because they cannot be studied directly in vivo. Human embryonic stem or induced pluripotent stem cells offer attractive in vitro alternatives to analyze the process. Here we review recent efforts to develop defined and quantitative systems to address outstanding developmental questions against a background of what we know about the development of hematopoietic cells in the fetus and derived from mouse embryonic stem cells.     

Human adenosine deaminase expression in mice

A replication defective retroviral vector containing a human adenosine deaminase (hADA) cDNA was produced by GP + E-86 packaging cells at high titer. We report long-term expression of hADA in the hematopoietic tissues of mice transplanted with bone marrow cells infected by in vitro co-cultivation with vector producing cells. Western analysis using an hADA-specific antibody allowed detection of the protein in the peripheral blood of all 37 transplanted mice for at least 9 weeks. Sixty-eight percent of the animals continued to express hADA in one or more of their hematopoietic tissues for the experimental period, and hADA was found in both spleen colonies and tissues of secondary recipients. There was provirus integration and expression in myeloid, erythroid, and lymphoid cell lineages, indicating extensive repopulation by the progeny of infected stem cells. The vector did not contain a selectable marker, and the infected stem cells did not have a competitive in vivo advantage. Nevertheless, we observed consistent gene transfer into hematopoietic stem cells and long-term expression of a human gene product in their progeny.     

Colony-forming ability of pluripotent hematopoietic stem cells in mice following tumor transplantation

The mechanism of tumor-induced hematological alterations at the level of pluripotent hematopoietic stem cells (CFU-S) was investigated in mice bearing transplantable ascites tumor, Sarcoma 180. Tumor growth for 10 days caused neutrophilic leukocytosis and decline in hemoglobin and RBC values in the peripheral blood, and significant reduction (p less than 0.05) in the concentration as well as absolute number of CFU-S in the femoral marrow but an increment in the spleen. Intraperitoneal administration of cell-free ascitic fluid caused similar alterations of CFU-S in normal mice, but heat-killed tumor cells failed to elicit such response. Tumor cell-conditioned medium when injected into normal mice caused CFU-S alterations in a pattern similar to that of tumor-bearing animals. It is concluded that alterations of CFU-S following tumor transplantation is attributable to tumor growth rather than the presence of dead or necrotic cells in the tumor inoculum. It is likely that tumor cells elaborate some factor(s) which mediate such changes.     

Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells.

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.     

Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.     

Retrovirus-mediated transfer of human adenosine deaminase gene sequences into cells in culture and into murine hematopoietic cells in vivo.

Deficiency of the enzyme adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4; ADA) leads to severe combined immunodeficiency, a disorder that potentially could be corrected by gene transfer into hematopoietic cells. We have constructed retroviruses containing human ADA cDNA and a dominant selectable marker, a mutated dihydrofolate reductase gene (DHFR*) encoding methotrexate resistance. Human ADA cDNA was inserted alone (DHFR*-ADA) or with a simian virus 40 (SV40) promoter (DHFR*-SVADA). Although NIH 3T3 cells infected with either construct produced human ADA activity, substantially greater levels were attained with DHFR*-SVADA. Infection of murine lymphoid cells in culture with DHFR*-SVADA led to expression of human enzyme at a level well above the mouse endogenous level. ADA activity was also increased after infection of a human ADA-deficient B-cell line. Lethally irradiated mice that were reconstituted with syngeneic marrow infected with the DHFR*-SVADA virus contained unrearranged, integrated proviral DNA in total spleen DNA or in spleen hematopoietic stem cell (CFU-S)-derived colonies. Nevertheless, no human ADA was detectable. RNA analysis showed relatively low and variable expression from the retroviral long terminal repeat, and no detectable expression from the internal SV40 promoter. These data suggest that intrinsic biologic differences exist between cultured cells and CFU-S in vivo.     

Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy.

Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli beta-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.     

Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a Transwell coculture system

The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus- producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.     

Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice

A recombinant AAV2 (rAAV2) vector encoding antisense RNA to HIV-1 transactivating region (TAR) was evaluated for transduction of human cord blood CD34+CD38- hematopoietic stem cells (HSC) capable of serial engraftment in nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Results revealed long-term multilineage marking in primary and secondary recipients, and significantly, an enrichment of transduced cells in secondary hosts, indicating efficient transduction of multipotential self-renewing HSC. These results were confirmed by the persistence of rAAV marking of clonogenic progenitors in serial analyses of recipient marrow. Upon HIV-1 challenge, the macrophage progeny of transduced CD34+ cells expressed antisense RNA and exhibited sustained and significant inhibition of virus replication as compared with controls in every donor tested, without selective pressure. This study represents a clear in vivo demonstration of efficient rAAV2 transduction of human HSC.     

Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34(+) cells and primary colony forming unit-cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34(+) cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P <.01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.     

Long-term expression of a T-cell receptor beta-chain gene in mice reconstituted with retrovirus-infected hematopoietic stem cells.

To determine the feasibility of retrovirus-mediated gene transfer into stem cells for studying T-cell development, we constructed a high-titer retrovirus vector containing the neomycin phosphotransferase (neo) gene and a murine T-cell receptor (TCR) beta-chain gene with the V beta 6 variable segment. The TCR gene was placed under the control of the human beta-actin promoter and enhancer. Bone marrow cells pretreated with 5-fluorouracil were infected by coculturing with psi-2 virus-producing cells in the presence of recombinant interleukins 1, 2, 4, and 6 as well as interleukin 3 from WEHI-3 conditioned medium. The infected cells were transplanted into irradiated mice, and expression of the exogenous V beta 6 gene was examined with a V beta 6-specific monoclonal antibody, RNase protection, and polymerase chain reaction amplification. Three of seven mice expressed the retroviral TCR gene on the surface of a significant proportion of mature T cells 5-6 months after transplantation. In mice analyzed less than 1 month after transplantation, up to 30% of mature T cells expressed V beta 6 TCRs, an increase of at least 20% above the level of endogenous V beta 6 expression. DNA analysis revealed that pluripotent hematopoietic stem cells were infected by the retroviral vector in a long-term reconstituted mouse that showed increased V beta 6 expression.