Michelle Millington

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34+ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin. Methodology/Principal Findings Using commercially available G-CSF mobilized peripheral blood (PB) CD34+ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin. Conclusions/Significance This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34+ cells.

CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape

Allogeneic transplantation with CCR5-delta 32 (CCR5-d32) homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC) to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN), clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9), transcription activator-like effectors nuclease (TALEN), short hairpin RNA (shRNA), and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.

Preclinical safety and efficacy of an anti-HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC). CCR5-targeted shRNA (sh5) and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

Promoter Targeting shRNA Suppresses HIV-1 Infection In vivo Through Transcriptional Gene Silencing

Despite prolonged and intensive application, combined antiretroviral therapy cannot eradicate human immunodeficiency virus (HIV)-1 because it is harbored as a latent infection, surviving for long periods of time. Alternative approaches are required to overcome the limitations of current therapy. We have been developing a short interfering RNA (siRNA) gene silencing approach. Certain siRNAs targeting promoter regions of genes induce transcriptional gene silencing. We previously reported substantial transcriptional gene silencing of HIV-1 replication by an siRNA targeting the HIV-1 promoter in vitro. In this study, we show that this siRNA, expressed as a short hairpin RNA (shRNA) (shPromA-JRFL) delivered by lentiviral transduction of human peripheral blood mononuclear cells (PBMCs), which are then used to reconstitute NOJ mice, is able to inhibit HIV-1 replication in vivo, whereas a three-base mismatched variant (shPromA-M2) does not. In shPromA-JRFL–treated mice, HIV-1 RNA in serum is significantly reduced, and the ratio of CD4+/CD8+ T cells is significantly elevated. Expression levels of the antisense RNA strand inversely correlates with HIV-1 RNA in serum. The silenced HIV-1 can be reactivated by T-cell activation in ex vivo cultures. HIV-1 suppression is not due to offtarget effects of shPromA-JRFL. These data provide “proof-of principle” that an shRNA targeting the HIV-1 promoter is able to suppress HIV-1 replication in vivo.

The use of cell-delivered gene therapy for the treatment of HIV/AIDS

HIV/AIDS is a disease that impairs immune function, primarily by decreasing T-lymphocyte count. Its progression can be contained by highly active antiretroviral therapy (HAART), but there are side effects that can be severe, and the development of resistance often forces the physician to modify the HAART regimen. There are no vaccines available for HIV. An alternative approach that could provide a path to a curative therapy is the use of cell-delivered gene therapy in which an anti-HIV gene(s) is introduced into hematopoietic cells to produce a population that is protected from the effects of HIV. In this paper, we review the field and discuss an approach using a short hairpin RNA to CCR5, an important co-receptor for HIV.

Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success

BackgroundMultiple short hairpin RNA (shRNA) gene therapy strategies are currently being investigated for treating viral diseases such as HIV-1. It is important to use several different shRNAs to prevent the emergence of treatment-resistant strains. However, there is evidence that repeated expression cassettes delivered via lentiviral vectors may be subject to recombination-mediated repeat deletion of 1 or more cassettes.ResultsThe aim of this study was to determine the frequency of deletion for 2 to 6 repeated shRNA cassettes and mathematically model the outcomes of different frequencies of deletion in gene therapy scenarios. We created 500+ clonal cell lines and found deletion frequencies ranging from 2 to 36% for most combinations. While the central positions were the most frequently deleted, there was no obvious correlation between the frequency or extent of deletion and the number of cassettes per combination. We modeled the progression of infection using combinations of 6 shRNAs with varying degrees of deletion. Our in silico modeling indicated that if at least half of the transduced cells retained 4 or more shRNAs, the percentage of cells harboring multiple-shRNA resistant viral strains could be suppressed to < 0.1% after 13 years. This scenario afforded a similar protection to all transduced cells containing the full complement of 6 shRNAs.ConclusionDeletion of repeated expression cassettes within lentiviral vectors of up to 6 shRNAs can be significant. However, our modeling showed that the deletion frequencies observed here for 6× shRNA combinations was low enough that the in vivo suppression of replication and escape mutants will likely still be effective.

A sensitive dual-fluorescence reporter system enables positive selection of ras suppressors by suppression of ras-induced apoptosis

We have developed a novel dual-fluorescence reporter system incorporating green (GFP) and red (RFP) fluorescent proteins to monitor expression of the N-rasm gene and an N-rasm suppressor, respectively. Retroviral vectors were produced in which human N-rasm (codon 13 mutation) was coexpressed with GFP, and a ribozyme specifically targeting N-rasm was coexpressed with RFP. N-Rasm suppression was monitored by measurement of GFP fluorescence in dual-fluorescent (GFP and RFP) cells. We demonstrated that the degree of N-rasm suppression was dependent on the ribozyme dose, proportional to red fluorescence, in dual-fluorescent cells. We further showed that ribozyme-mediated N-rasmsuppression inhibited growth of NIH3T3 and CD34-positive TF-1 cells. In these cultures, ras suppressor activity resulted in the depletion of suppressor-positive cells due to inhibition of cell growth. In contrast, N-rasm suppression produced a growth advantage to human leukemic K562 cells, presumably by inhibiting N-rasm-induced apoptosis. In K562 cells, ras suppression resulted in the outgrowth of suppressor-positive cells. This provides a platform to identify suppressors of ras that is based on function.

V-myc in a simple, single gene retroviral vector causes rapid induction of leukemia and concomitant apoptosis following bone marrow transplantation.

We have previously developed an in vivo model of leukemogenesis utilizing mice reconstituted with genetically modified bone marrow cells. Based on those studies, a new single gene retroviral vector has been engineered which efficiently transfers v-myc into immature murine bone marrow cells. All reconstituted mice developed leukemia with a short latency period (5–11 weeks). In addition to hyperproliferation associated with elevated levels of PCNA, extensive apoptosis was also observed in all leukemic animals with p53 accumulating in the apoptotic cells. Whereas bax encoded protein, an effector of p53 apoptotic activity was detected in apoptotic cells, p21Waf1 protein, a potential mediator of p53 growth suppression was not detected in these cells suggesting that v-myc-induced apoptosis was independent of the ability of p53 to induce p21Waf1. These results indicate that apoptosis, a part of the cellular response to v-myc expression, does not prevent leukemia development and that hyperproliferation rather than abrogation of oncogene-induced apoptosis appears to be a critical event in v-myc-induced leukemia.

Induced p21WAF1 expression acts to reverse myc myelomonocytic cell transformation.

Two murine myelomonocytic cells lines were used to examine p21WAF1 expression in myc-induced cell transformation. tEMmyc4 and FDLV are two v-myc–transformed immortalised myeloid cell lines exhibiting different transformed phenotypes. FDLV cells were derived from the transduction of v-myc into FDC-P1 cells and retain growth factor (IL-3) dependence, whereas tEMmyc4 cells were derived from the transduction of embryonal monocytes with v-myc and are growth factor–independent, constitutively express endogenous CSF-1, and are highly tumorigenic in syngeneic mice. Both cell lines were found to exhibit low p21WAF1 expression. When examined in tEMmyc4 cells, neither the p53-dependent pathway (mitomycin C or exogenous p53) nor p53-independent pathway (TPA or growth factor, CSF-1, stimulation) acted to increase p21WAF1 levels. Growth factor (IL-3) withdrawal, shown to reduce p21WAF1 levels in parental FDC-P1 cells, failed to do this in FDLV cells. The dependence of p21WAF1 expression on v-myc was further demonstrated by showing that a v-myc–targeted ribozyme, which acts to decrease v-myc RNA, increased p21WAF1 levels in tEMmyc4 cells. Enforced expression of exogenous p21WAF1 in tEMmyc4 cells with dysfunctional growth cycle (including growth arrest and increased susceptibility to apoptosis) was examined. p21WAF1 partially restored cell cycle regulation and apoptosis as well as inhibited the delayed cell cycle progression and apoptosis induced by mitomycin C or serum withdrawal. These results show p21WAF1 expression to be affected by v-myc and a restoration of p21WAF1 expression to partially reverse myc-mediated transformation.Cancer Gene Therapy (2000) 7, 1491–1503

The β-Galactosidase Gene as a Marker for Hematopoietic Reconstitution: Individual Cell Analysis in a Murine Bone Marrow Transplantation Model

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.