Victor Bartsevich

Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases

Homozygosity for the naturally occurring Δ32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted ∼50% of CCR5 alleles in a pool of primary human CD4+ T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4+ T cells had lower viral loads and higher CD4+ T-cell counts than mice engrafted with wild-type CD4+ T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4+ T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN–modified autologous CD4+ T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.

Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome

Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a safe harbor locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.

Engineered zinc finger proteins for controlling stem cell fate.

Stem cells are functionally defined as progenitor cells that can self‐renew and differentiate. Critical transitions in these cells are controlled via signaling pathways and subsequent transcriptional regulation. Technologies capable of modulating the levels of gene expression, especially those of transcription factors, represent powerful tools for research and could potentially be used in therapeutic applications. In this study, we evaluated the ability of synthetic zinc finger protein transcription factors (ZFP‐TFs) to cause the differentiation of embryonic stem (ES) cells. We constructed ZFP‐TFs that target the mouse Oct‐4 gene (which is a major regulator of ES cell pluripotency and self‐renewal). These designed transcription factors were able to regulate the transcription of Oct‐4, affecting the expression of downstream genes and thus regulating ES cell differentiation.

Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol

Clinical translation of in vivo genome editing to treat human genetic diseases requires thorough preclinical studies in relevant animal models to assess safety and efficacy. A promising approach to treat hypercholesterolemia is inactivating the secreted protein PCSK9, an antagonist of the LDL receptor. Here we show that single infusions in six non-human primates of adeno-associated virus vector expressing an engineered meganuclease targeting PCSK9 results in dose-dependent disruption of PCSK9 in liver, as well as a stable reduction in circulating PCSK9 and serum cholesterol. Animals experienced transient, asymptomatic elevations of serum transaminases owing to the formation of T cells against the transgene product. Vector DNA and meganuclease expression declined rapidly, leaving stable populations of genome-edited hepatocytes. A second-generation PCSK9-specific meganuclease showed reduced off-target cleavage. These studies demonstrate efficient, physiologically relevant in vivo editing in non-human primates, and highlight safety considerations for clinical translation.

200. Generation of CAR-T Cells Lacking T Cell Receptor and Human Leukocyte Antigen Using Engineered Meganucleases

The manufacture of CAR-T cells depends on peripheral blood donations that contain T cells of sufficient quality and quantity. Currently, many CAR-T programs rely on autologous T cells, but several technical and commercial challenges hinder development. The majority of CAR-T trials have enrolled leukemia or lymphoma patients, many of which are unsuitable donors for CAR-T production due to their disease state or to previous treatments with lymphodepleting agents. In addition, a custom CAR-T production run for each patient is time consuming, lacks standardization and may present regulatory challenges. An alternative strategy is to source T cells from healthy donors and produce large batches of allogeneic CAR-T cells. Allogeneic T cells, however, will display mismatched human leukocyte antigens (HLA) that will be recognized by the recipients’ immune systems, contributing to immune rejection of engrafted CAR-T cells. Additionally, donor T cells will recognize the mismatched HLAs present in the recipient, contributing to graft-versus-host immune pathology. Both undesired immune responses are predicated on interactions between HLA and T cell receptors (TCR), and while the therapeutic effectiveness of CAR-T cells with targeted deletions in TCR genes has been reported by several groups, studies featuring both TCR and HLA deletion are limited. Here, we describe the use of meganucleases engineered to target regions of the TCR α chain constant region and β-2 microglobulin genes to generate TCR and HLA class I knockout primary human T cells. Both nucleases generate knockouts with approximately 75% efficiency and are well-tolerated by primary T cells from at least four separate donors. Purified double knockout cells do not demonstrate functional disadvantages in terms of proliferation or cytokine production, but do exhibit reduced allostimulatory potential toward HLA-mismatched T cells. Together, these findings demonstrate the feasibility of generating therapeutic quantities of CAR-T cells with reduced allo-reactive potential and collateral toxicity to normal tissues in recipients.

138. Potential Therapeutic Treatment of Friedrich's Ataxia Using Highly Specific Engineered Meganucleases

Friedreichs ataxia (FRDA) is a recessive genetic disorder that results in progressive neuromuscular deterioration. The most common causative genetic alteration of this disorder is an expansion of a trinucleotide (GAA/TTC) repeat within the first intron of the Frataxin (FXN) gene. This triplet expansion causes transcriptional defects, resulting in low FXN mRNA and protein levels. In patients, the correlation between number of repeats and severity of the disease, age of onset and cardiomyopathy indicates that the repeat expansion is the primary cause of FRDA, thus making it a potential therapeutic target. We are investigating the potential use of our engineered meganuclease technology to precisely remove these trinucleotide repeats as a therapeutic treatment for FRDA. We have designed a pair of meganucleases that introduce double-strand breaks on either side of the FXN trinucleotide repeat region, generating compatible 3’ overhangs that can be repaired by direct ligation, resulting in precise excision of the intervening region. Introduction of this pair of meganucleases into FRDA patient fibroblasts in cell culture results in the successful deletion of the causative repeat region in greater than 15% of the transfected cells. This removal of the FRDA repeat region has been confirmed using both digital PCR-based analytics and deep sequencing. Additional analysis of Frataxin mRNA production, Frataxin protein levels, and cellular metabolism will further demonstrate the utility of the repeat excision approach for FRDA gene therapy.

51. A Novel Approach to Stem Cell Gene Therapy: Controlling Stem Cell ifferentiation with Engineered Zinc-Finger Protein Transcription Factors

Stem cells hold tremendous promise for gene therapy, tissue engineering, and the treatment of a diverse range of injuries and disease. However, exploiting the full potential of these pluripotent progenitor cells requires the establishment of a new set of tools capable of controlling the molecular decisions that determine whether, and how these cells differentiate. Critical transitions in stem cells are controlled via signaling pathways and subsequent transcriptional regulation. In this regard, we have previously shown that designed zinc-finger protein transcription factors (ZFP TFs) are capable of regulating the expression of targeted endogenous genes with singular specificity. In the present study, we evaluated the ability of such designed ZFP TFs to control the differentiation of embryonic stem (ES) cells. To this end, we constructed ZFP TFs that target the mouse OCT4 gene, a major regulator of ES cell pluripotency and self-renewal. We show here that introduction of an activator version of this ZFP TF resulted in increased OCT4 mRNA, and the expected concomitant effects on the expression of OCT4 responsive downstream target genes. Conversely, a repressor version of the OCT4-specific ZFP TF caused a robust reduction in OCT4 expression and the reciprocal downstream effects. Significantly, ZFP TF-dependent morphological differentiation of ES cells was also observed in a manner specific to the effector domain employed. Thus ZFP TFs are shown to be capable of controlling ES cell differentiation, raising the potential for their broad application to stem cell-based therapies.

302. Engineered Zinc Finger Transcription Factors: A Platform for Therapeutic Gene Regulation with Exquisite Specificity

A challenge of modern medicine is the development of effective yet highly specific drugs. To this end, we are developing artificial zinc finger protein transcription factors (ZFP TFs) as direct therapeutic agents for the treatment of disease. Here we show that ZFP TFs can be designed to activate or repress a wide range of clinically relevant genes, including Phospholamban, a regulator of heart contractivity, Chk2, a kinase with a role in DNA damage pathway and P2X7, a ligand-gated channel. Moreover, using these examples and the Affymetrix GeneChipR platform we show that these ZFP TFs regulate the targeted gene with exceptional and often singular specificity genome wide, despite the complexity of mammalian genome. This exquisite specificity is independent of both gene target and species, while occurring in the context of maximal efficacy. This combination of potency and specificity supports the clinical potential of ZFP TF technology.