RESUMO
The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5(Δ32/Δ32)) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5(Δ32/Δ32) donors, we reasoned that engineered autologous CD34(+) hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.
Assuntos
Endonucleases/genética , Genômica/métodos , Células-Tronco Hematopoéticas/citologia , Receptores CCR5/genética , Dedos de Zinco/genética , Síndrome da Imunodeficiência Adquirida/terapia , Adenoviridae/genética , Animais , Antígenos CD34/genética , Antígenos CD34/metabolismo , Apoptose , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Endonucleases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Deleção de Genes , Marcação de Genes , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Receptores CCR5/metabolismoRESUMO
Genetic strategies to block expression of CCR5, the major co-receptor of human immunodeficiency virus type 1 (HIV-1), are being developed as anti-HIV therapies. For example, human hematopoietic stem/precursor cells (HSPC) can be modified by the transient expression of CCR5-targeted zinc finger nucleases (ZFNs) to generate CCR5-negative cells, which could then give rise to HIV-resistant mature CD4(+) T cells following transplantation into patients. The safety and anti-HIV effects of such treatments can be evaluated by transplanting ZFN-treated HSPC into immunodeficient mice, where the extent of human cell engraftment, lineage differentiation and anti-HIV activity arising from the engineered HSPC can be examined. In this way, humanized mice are providing a powerful small animal model for pre-clinical studies of novel anti-HIV therapies.
Assuntos
Terapia Biológica/métodos , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/fisiologia , Células-Tronco Hematopoéticas/virologia , Receptores CCR5/genética , Receptores de HIV/genética , Animais , Terapia Biológica/efeitos adversos , Humanos , Camundongos , Camundongos SCID , Receptores CCR5/deficiência , Receptores de HIV/deficiência , Dedos de ZincoRESUMO
Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).