Lentivirus-mediated gene transfer into hematopoietic repopulating cells in baboons

Efficient transduction of hematopoietic stem cells is a prerequisite for successful hematopoietic stem cell gene therapy. Oncoretroviral vectors are the most widely used vectors for hematopoietic gene therapy studies. However, these vectors require cell division, and thus efficient transduction of quiescent stem cells has been difficult to achieve. Lentiviral vectors can transduce non-dividing cells and therefore may be more efficient in transducing quiescent hematopoietic stem cells. We have used a competitive repopulation assay in the baboon to compare transduction of hematopoietic repopulating cells by lentiviral and oncoretroviral vectors. Baboon CD34-enriched marrow cells were transduced in the presence or absence of multiple hematopoietic growth factors using a short, 2-day, transduction protocol. Here, we show that efficient lentiviral transduction of hematopoietic repopulating cells was only achieved when cells were transduced in the presence of multiple growth factors. Using these conditions, up to 8.6% of hematopoietic repopulating cells were genetically modified by the lentiviral vector more than 1 year after transplant. Interestingly, the number of lentivirally marked cells increased over time in three of four animals. In conclusion, these results suggest that lentiviral vectors are able to tranduce multilineage hematopoietic stem cells, and thus, may provide an alternative vector system for clinical stem cell gene therapy applications.     

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector

Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34(+) cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34(+) cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34(+) cells transduced with the Ad35 vectors was 12-76 and 1.4-3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34(+) cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34(+) cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs.     

Efficient transduction and engraftment of G-CSF-mobilized peripheral blood CD34+ cells in nonhuman primates using GALV-pseudotyped gammaretroviral vectors.

The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large-animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF); however, SCF is unavailable for clinical use in the United States and the European Union. A recent study in a competitive repopulation assay in the rhesus macaque showed very inefficient marking of G-CSF-mobilized (G/only) peripheral blood (G-PBSC) CD34(+) cells relative to G-CSF and SCF-mobilized cells using vectors with an amphotropic pseudotype. Because G-PBSC would be the preferred target cell population for most clinical stem cell gene therapy applications, we asked whether we could achieve efficient transduction and engraftment of G-PBSC using Phoenix-GALV-pseudotyped vectors. We transplanted three baboons with G/only mobilized CD34(+) cells transduced with GALV-pseudotyped retroviral vectors. We observed high-level, persistent engraftment of gene-modified G-PBSC in all animals with gene marking levels in granulocytes up to 60%. We analyzed amphotropic (PIT2) and GALV (PIT1) receptor expression in G/only cells and found preferential expression of PIT1 after G/only, which may explain the inferior results with amphotropic pseudotypes. These findings demonstrate that high stem cell gene transfer levels can be achieved using G-CSF-mobilized PBSC with Phoenix-GALV-pseudotyped vectors.     

Progress in the use of gene transfer methods to treat genetic blood diseases

A report by French physician-scientists suggests a successful application of gene transfer methods in the treatment of two children with severe combined immunodeficiency (SCID) due to defective interleukin 2 receptor common gamma chain. The protocol used in this clinical trial was derived from a number of preclinical and basic studies leading to improved transduction of hematopoietic stem and primitive progenitor cells using retrovirus vectors. These improvements have also been shown to impact transduction of a long-lived progenitor cell in a chemotherapy protocol in cancer patients. The improved results of these human trials come during a period of increased scrutiny and criticism of human gene therapy trials, due, in part, to significant toxicities in some trials using adenovirus-based vectors. The potential efficacy versus toxicity of phase I trials of human gene therapy is also under question. After many years of research, however, there appears to be real evidence that genetic diseases may be successfully treated by gene transfer techniques. Future clinical studies should be based on continued progress in the understanding of the toxicology of gene delivery systems, vector technology, and target cell manipulation.     

Transduction of long-term and mobilized peripheral blood-derived NOD/SCID repopulating cells by foamy virus vectors

Foamy virus (FV) vectors are a promising gene delivery system for use in hematopoietic stem cell gene therapy. Previous FV vector marking studies in the NOD/SCID xenotransplantation model used umbilical cord blood (UCB)-derived SCID repopulating cells (SRCs) that were assayed 5-10 weeks posttransplantation. We now report efficient FV vector transduction (>65%) of UCB-derived primitive, long-term SRCs engrafted for 18 weeks. In addition, we evaluated gene transfer into mobilized peripheral blood (MPB)-derived SRCs by improved, deleted FV vectors containing minimal cis-acting sequences and packaged by split helper constructs that would be appropriate for use in clinical trials. When used at a multiplicity of infection of 1 in a 10-hr transduction protocol, these improved vectors transduced 34% of engrafted MPB-derived SRCs.     

Silencing p21(Waf1/Cip1/Sdi1) expression increases gene transduction efficiency in primitive human hematopoietic cells

Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors, which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here, we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA, synthesized siRNA and transcribed shRNA, reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction, however, was sufficient to increase transduction efficiency to two- to four-fold in cell cultures, and followed by a seven- to eight-fold increase in mice. The RNAi treated, lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together, both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus, RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy.     

Alternative viral envelopes for oncoretroviruses to increase gene transfer into hematopoietic stem cells

The hematopoietic stem cell is the target for gene therapy of human blood disease. Low retroviral receptors for the commonly used vectors and quiescence of hematopoietic stem cells are believed to be major obstacles to the success of gene therapy. The development of new stem cell assays has allowed better understanding of the biology and phenotype of hematopoietic stem cells, leading to selection of highly enriched populations of hematopoietic stem cells. Quantitation of retrovirus receptors on these enriched populations of hematopoietic stem cells has resulted in the identification of subpopulations of cells expressing high levels of retrovirus receptors. New promising retrovirus envelopes are being developed. In this review, we discuss those issues that may help to resolve the problem of low gene transfer efficiency into human hematopoietic stem cells.     

Use of nonintegrating lentiviral vectors for gene therapy

Vectors based on lentiviruses have become potent tools for efficient gene transfer to multiple cell types both in vitro and in vivo. In part this is attributable to the stability of transduction afforded by integration into the target cell genome. However, evidence indicates that episomal forms of the vector can also be harnessed for effective gene expression. Nonintegrating vectors retain the high transduction efficiency and broad tropism of conventional lentiviruses but avoid the potential problems associated with the nonspecific integration of a transgene. In this respect they are particularly useful in postmitotic tissue because the vector genome is not diluted out through cell division. Here we discuss the various mutations that may be introduced into human immunodeficiency virus-based lentiviral vectors to achieve efficient transduction, and the mechanisms by which these vectors are effective. We also discuss their potential application to gene therapy and the treatment of genetic disease.     

G2 cell cycle arrest and cyclophilin A in lentiviral gene transfer.

Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) have a higher propensity to transduce nondividing cells compared to vectors based on oncoretroviruses. We report here that genistein, a previously known protein tyrosine kinase (PTK) inhibitor and G2 cell cycle arrest inducer, significantly enhanced lentiviral transduction in a dose-dependent manner. Increased transduction, as measured by vector expression, was seen in a variety of human cell lines, murine primary lymphocytes, and primary human CD34(+) peripheral blood progenitor cells as well. Increased vector expression was also associated with an increase in vector DNA copy number, as assessed by quantitative PCR. Genistein-mediated G2 cell cycle arrest, rather than PTK inhibition, appears to be the major factor responsible for increased gene transfer. Genistein also increases cyclophilin A (CypA) protein, a cellular protein important for efficient HIV-1 infection. While we show that CypA(-/-) Jurkat cells transduce poorly with lentiviral vectors, genistein does increase gene transfer in CypA-deficient cells. CypA and G2 cell cycle arrest appear to be two independent factors important for efficient lentiviral gene transfer. The role of genistein and other G2-arresting agents may be useful for improving the efficiency of lentiviral gene therapy.     

Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo

Low efficiencies of nonviral gene vectors, the receptor-dependent host tropism of adenoviral or low titers of retroviral vectors limit their utility in gene therapy. To overcome these deficiencies, we associated gene vectors with superparamagnetic nanoparticles and targeted gene delivery by application of a magnetic field. This potentiated the efficacy of any vector up to several hundred-fold, allowed reduction of the duration of gene delivery to minutes, extended the host tropism of adenoviral vectors to nonpermissive cells and compensated for low retroviral titer. More importantly, the high transduction efficiency observed in vitro was reproduced in vivo with magnetic field-guided local transfection in the gastrointestinal tract and in blood vessels. Magnetofection provides a novel tool for high throughput gene screening in vitro and can help to overcome fundamental limitations to gene therapy in vivo.     

A new direction for gene therapy: intrathymic T cell-specific lentiviral gene transfer

Reports of neoplasia related to insertional activation of protooncogenes by retroviral vectors have raised serious safety concerns in the field of gene therapy. Modification of current approaches is urgently required to minimize the deleterious consequences of insertional mutagenesis. In this issue of the JCI, Adjali and colleagues report on their treatment of SCID mice lacking the 70-kDa protein tyrosine kinase, ZAP-70, with direct intrathymic injection of a ZAP-70-expressing T cell-specific lentiviral vector, which resulted in T cell reconstitution. Using lentiviral vectors and in situ gene transfer may represent a safer approach than using retroviral vectors for ex vivo gene transfer into HSCs, avoiding 3 factors potentially linked to leukemogenesis, namely HSC targets, ex vivo transduction and expansion, and standard Moloney leukemia virus-based retroviral vectors.     

Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral- mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.     

Green fluorescent protein as a selectable marker of fibronectin-facilitated retroviral gene transfer in primary human T lymphocytes

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on markers that can be rapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent protein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stable fluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP system to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions of primary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell line in which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV) envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were transduced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistently correlate with enhanced gene transfer, indicating that factors other than proliferation are important for optimal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T cell transduction and will enable further optimization of T cell gene therapy protocols.     

Primary T lymphocytes as targets for gene therapy

Peripheral blood T lymphocytes have been considered an attractive target for gene therapy applications. They can be easily harvested and readily expanded ex vivo. The transduction efficiency of primary human lymphocytes with standard retroviral vectors approaches 50% or more using optimized methods of gene transfer. Other methods of gene transfer, including adenoviral, adeno-associated viral, and lentiviral vectors, or nonviral techniques, have also been used for gene transfer into primary lymphocytes. Despite encouraging results in vitro, human clinical trials using retroviral vectors to transduce primary lymphocytes have been hindered by low expression levels of transgenes and immune responses against transgene products. Strategies to overcome these problems need to be developed.     

Nonviral gene delivery with the sleeping beauty transposon system

Effective gene therapy requires robust delivery of therapeutic genes into relevant target cells, long-term gene expression, and minimal risks of secondary effects. Nonviral gene transfer approaches typically result in only short-lived transgene expression in primary cells, because of the lack of nuclear maintenance of the vector over several rounds of cell division. The development of efficient and safe nonviral vectors armed with an integrating feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was shown to provide efficient stable gene transfer and sustained transgene expression in primary cell types, including human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced pluripotent stem cells, and T cells. These cells are relevant targets for stem cell biology, regenerative medicine, and gene- and cell-based therapies of complex genetic diseases. Moreover, the first-in-human clinical trial has been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. We discuss aspects of cellular delivery of the SB transposon system, transgene expression provided by integrated transposon vectors, target site selection of the transposon vectors, and potential risks associated with random genomic insertion.     

Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.

The improvement of initial tumor cell transduction with viral vectors is a major task in tumor gene therapy. We have developed mouse tumor models with hepatic metastases to study transduction of tumor cells after systemic adenovirus vector application. The tumor models were established by intraportal transplantation of human tumor cell lines into immunodeficient mice. Liver metastases derived from cervix, colon, breast, and liver cancer lines were analyzed for distribution of extracellular matrix, vascularization, and transgene expression after tail vein injection of adenovirus vectors. Overall, xenografts resembled the morphology of corresponding tumors in cancer patients. Adenovirus-mediated gene delivery depended on tumor vascularization and direct contact between blood vessels and tumor cells. These models represent important tools for studying and improving tumor gene therapy approaches.     

Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene

The low transduction efficiency of viral and nonviral vectors is a major limitation in tumour gene therapy. The HSV-1 tegument protein VP22 has been shown to exhibit a novel intercellular transport property. VP22 wild-type as well as VP22 fusion proteins efficiently spread from the original expressing cell to numerous neighbouring cells, so that protein transport by VP22 chimaeric polypeptides into the surrounding cells offers a possible compensation for the inadequate gene transfer efficiencies. To improve the therapeutic efficacy of the E. coli cytosine deaminase (CD) suicide gene we made use of the VP22 transport property in CD transducing adenoviral (Ad) vectors. C- and N-terminal fusions of CD linked in-frame with VP22 were generated and cloned into recombinant adenoviral vectors. Following in vitro transduction immunofluorescence analysis of Ad-transduced producer cells coplated with naive cells confirmed that the characteristic foci pattern of central producer and adjoining neighbour cells displaying nuclear staining was retained. After transduction of rat hepatoma cells with adenoviral vectors and subsequent incubation with the prodrug 5-FC, we observed enhanced cell cytotoxicity when comparing the CD-VP22 fusion (Ad-CD-VP22) with Ad-vectors expressing the CD gene only (Ad-CD). Thereby employment of Ad-vectors encoding VP22 fusion proteins opens up new possibilities to potentiate the efficiency of suicide gene therapy for the treatment of solid tumours.