Malika Hale

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

Therapeutic coding sequences can be targeted to the CCR5 locus of primary human T cells with high efficiency by using megaTAL nuclease and an AAV donor template. Delete and replace Newer gene-editing methods hold promise for correcting human disease but so far have been hampered by low efficiencies when used in primary cells. To address this issue, Sather et al. have devised a more effective way to both disrupt and replace the CCR5 locus in human T cells, a procedure that has already been shown to improve HIV clearance. Serotype 6 of an adeno-associated viral vector worked particularly well for delivery of megaTAL nucleases and homologous donor templates to primary human T cells, achieving efficient gene-editing rates and little toxicity. The megaTALs generate homology-directed repair (rather than previous efforts, which induce nonhomologous end-joining repair) and so was used for both deletion and accurate replacement of the CCR5 locus. The authors demonstrate that chimeric antigen receptors and an HIV fusion inhibitor inserted into the CCR5 locus ameliorate HIV infection in mice and show that their approach also works in CD34+ hematopoietic precursor cells. Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4+ T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)–mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP–modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34+ cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patients immune system and provide protection from HIV infection.

Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells

The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.

761. Targeted Killing of HIV Infected Cells Using CCR5-Disrupted Anti-HIV-CAR T Cells

A cure for HIV remains an important treatment goal for 30 million HIV-infected individuals worldwide. Long term control of HIV following a single treatment will require a mechanism to eradicate the latent reservoir of HIV infected cells that are capable of reactivating. A previous phase II clinical trial using an anti-HIV chimeric antigen receptor (αHIV-CAR) targeting the CD4-binding site on HIV envelope was partially effective. To optimize this approach we have developed a series of CARs based on the scFV of broadly neutralizing HIV antibodies targeting four different structural regions of the HIV envelope: the V1/V2 loop, the V3 loop, the CD4 binding site, and the membrane-proximal external region. αHIV-CAR T cells targeting different epitopes were compared and were able to kill > 80% of HIV-infected cells grown in the presence of ART. However, a limitation of αHIV-CAR T cells is that the αHIV-CAR also serves as a receptor for HIV and allows HIV infection of αHIV-CAR T cells. Therefore we disrupted the major co-receptor required for HIV cell entry, CCR5, using a megaTAL nuclease, as a means of protecting the CAR-expressing cells from HIV infection. We used two strategies for achieving these dual modifications in primary human T cells. For both strategies, the CCR5 megaTAL nuclease was delivered by mRNA electroporation, which has previously been shown to induce a high rate of bi-allelic NHEJ-mediated gene disruption. αHIV-CAR expression cassettes were delivered into the host genome via lentiviral vectors (LV), or were targeted to the megaTAL nuclease cleavage site in CCR5 using an adeno-associated virus (AAV) that included CCR5 homology arms. Both strategies resulted in stable expression of the CAR construct and specific activation of CAR+ T cells in the presence of an HIV+ cell line. In the presence of actively replicating virus, CCR5-megaTAL treated CAR+ T cells out-performed CAR+ T cells generated by LV delivery alone as measured by reduction in HIV capsid protein. To enable testing in non-human primates (NHP), we re-optimized the cell-editing protocol with NHP lymphocytes. Primary T cells from pigtail macaques were successfully transfected with CCR5 megaTAL mRNA and achieved a CCR5 disruption rate of 50%. Cells were subsequently transduced with αHIV-CAR LV resulting in ~70% of manipulated cells with αHIV-CAR. Expansion of the cells in vitro resulted in 60-fold expansion over 8 days. CAR+ NHP cells specifically killed HIV infected human cells demonstrating that NHP-derived αHIV-CAR+ T cells retained killing function. In conclusion, it is feasible to construct αHIV-CAR+ T cells that are protected from HIV infection in human and NHP cells, and warrants further study in vivo.

238. BTK-Promoter LV Vectors Utilizing Conserved Intron Element Mediate Functional Rescue in Murine XLA

X-linked agammaglobulinema (XLA) is a rare immunodeficiency disorder caused by a mutation in the gene coding Brutons tryosine kinase (BTK). BTK is required for signal transduction downstream of the B cell antigen receptor (BCR) and is essential for normal B cell development, activation, and survival. Patients lacking BTK have very few mature B cells, markedly reduced antibody production, and suffer from recurrent and life-threatening infections. XLA is an ideal candidate for hematopoietic gene therapy, as B cells that express BTK are positively selected during development. We previously developed a SIN-lentiviral (LV) construct that used the human BTK promoter (BTKp) to drive BTK expression. We also incorporated a truncated, 0.7 kb, fragment derived from the HNRPA2B1/CBX3 ubiquitously acting chromatin opening element (UCOE) upstream of the BTKp to facilitate stable BTK expression over time. Bone marrow transplantation in a murine XLA model (BTK-/-Tec-/- mice) using LV transduced hematopoietic stem cells (HSCs) reconstituted myeloid and B cell numbers, BCR-dependent B cell proliferation and T-dependent antibody responses. The goal of the current study was to further optimize BTK expression in B and myeloid cells while reducing the viral copy number (VCN) required for efficient functional reconstitution. Using the ENCODE database, we identified several highly conserved upstream and intronic regions exhibiting histone modifications and transcription factor binding profiles in B cells consistent with enhancer regions. These conserved elements were introduced individually or in tandem into the LV construct between the UCOE and BTKp and tested for their ability to rescue BTK expression and B cell development in the murine XLA model. Addition of two key intronic regions led to consistent BTK expression and rescued the development and function of B cells, despite a >5-fold reduction in VCN per splenic B cell. Our findings illustrate the usefulness of this strategy for improving the efficacy of endogenous promoter-based genetic therapies. This new LV vector, UCOE. I4-5.BTKpro-hBTK, is currently being evaluated using mobilized CD34+ stem cells isolated from XLA patients and in additional pretoxicology studies designed to support its application in a future LV clinical trial for XLA.

284. High-Efficiency Targeted Introduction of an Anti-CD19 CAR Into the CCR5 Locus in Primary Human T Cells

The safety and efficacy of anti-CD19 chimeric antigen receptor (CD19-CAR) therapeutic cell transfer for treatment of CD19+ B cell malignancies is currently being evaluated in multiple clinical trials. Current protocols employ randomly integrating lentiviral or gamma-retroviral vectors to introduce the CAR into patient- or donor-derived T cells. This strategy carries with it an inherent risk of insertional mutagenesis as well as variable CAR expression. Here we have developed a method of targeting a CD19-CAR construct directly into the CCR5 locus by homology directed recombination (HDR). Using a CCR5 megaTAL nuclease (an engineered homing endonuclease and TALEN chimera) and an AAV donor template (comprised of an MND promoter-CD19CAR-T2A-BFP expression cassette between CCR5 homology arms), we obtained efficient targeting rates in primary human T cells. CAR expression was confirmed by flow cytometry and homologous recombination within the CCR5 locus was verified by PCR analysis and by direct sequencing. Rates of biallelic HDR were assessed using single cell PCR. T cells with the CD19-CAR at the CCR5 locus demonstrated activation, cytokine production and specific killing in the presence of CD19+ cells. Activity levels were indistinguishable from T cells transduced with LV expressing an identical CD19-CAR construct. Seamless targeting to CCR5 has important therapeutic advantages over traditional viral delivery, including a reduced potential for oncogenic insertions and uniform levels of gene expression, in parallel with effective disruption of the co-receptor for CCR5-tropic HIV-1. Further, the targeting methods used are directly amenable to clinical application. As HIV patients develop aggressive B cell lymphomas at increased rates, we believe this technique has substantial potential for clinical implementation including the protection of autologous CD19-CAR effector T cell products from HIV infection in vivo.

452. MegaTAL Disruption of CCR5 To Protect Anti-HIV CAR+ Lymphocytes from HIV Infection

More than 30 million people are infected with HIV. Antiretroviral therapy (ART) dramatically decreases mortality, but HIV-infected individuals on ART have an increased risk of malignancies, cardiovascular disease, neurologic disease, and shortened life expectancy. Therefore a cure for HIV remains an important treatment goal. A previous Phase II randomized clinical trial of anti-HIV Chimeric antigen receptor (CAR)-expressing T-cells was partially effective. We hypothesize that a limitation of the previous strategy was that the anti-HIV CAR+ lymphocytes were susceptible to HIV infection.

685. Efficient Targeted Gene Modification in Primary Human Hematopoietic Cells Using Co-Delivery of Nuclease mRNA and AAV Donors

Current clinical HIV treatment strategies applying gene-editing technologies to disrupt the HIV co-receptor CCR5 via nuclease delivery are limited by inefficient bi-allelic disruption and the lack of a combinatorial approach to control HIV infection. The ability to target additional factors conferring HIV resistance to the CCR5 locus with efficient bi-allelic disruption has the potential to translate to improved clinical outcomes. Here we demonstrate a novel method in which RNA-based nuclease expression is paired with AAV-mediated delivery of a homologous gene-targeting donor template to achieve highly efficient targeted recombination in primary human T-cells. This method consistently achieves an average of 60% rates of homology directed recombination into the CCR5 locus in T-cells (>20 experiments with multiple donors); with up to 90% of the gene-modified population exhibiting bi-allelic modification. T-cells modified with this protocol maintain a diverse repertoire and engraft in immune deficient, NOD/SCID/gc-/- (NSG) mice as efficiently as unmodified control T-cells. Using this method, we have integrated chimeric antigen receptors (CARs) into the CCR5 locus; and show that the resulting “targeted CAR” or tCAR T-cells exhibit anti-tumor activities indistinguishable from those generated using lentiviral random integration. Alternatively, we introduced the C46 HIV fusion inhibitor by targeted recombination generating T cell populations with very high-rates of bi-allelic CCR5 disruption paired with protection from HIV with CXCR4 co-receptor tropism. Next, as proof of principle, we utilized the CCR5 site as a ‘safe harbor’ locus for targeted gene addition of the human Wiskott-Aldrich syndrome (WAS) gene. Finally, this novel protocol was also successfully applied to adult human mobilized CD34+ cells, resulting in 10-20% homologous gene targeting, the first such report in adult human CD34+ cells at the CCR5 locus. Our results demonstrate that high-efficiency targeted integration is feasible in primary human cells, and highlight the potential of gene editing technologies to engineer CAR T-cells or other T cell products with myriad novel functional properties.

116. MegaTAL Nucleases Outperform TALENs in Promoting Homology-Directed-Gene Modification in Primary Human T Cells

Advances in rare cleaving nuclease technologies have dramatically increased the potential for seamless manipulation of virtually any gene (i.e. gene editing). Among these platforms, TALENs and Cas9-based nucleases have become broadly applied owing to ease in customizing their DNA recognition properties. In contrast, LAGLIDADG homing endonucleases (LHE) are more challenging to engineer, yet exhibit superior physical attributes, DNA recognition and hydrolysis properties. Recent advances have overcome hurdles in LHE engineering leading to the ability to more rapidly customize LHEs as therapeutic nucleases. In addition, LHEs can be fused to TAL effector arrays to create chimeric proteins [referred to here as megaTALs (MTAL)] that exhibit further enhancements in activity. We previously developed a CCR5-specific MTAL as a potential HIV therapeutic. In parallel, we generated high-activity TALEN pairs targeting the identical region in CCR5. In the current study, we directly compared the ability of these nuclease platforms to induce homology directed repair (HDR) in primary human T-cells. We first verified that both reagents facilitated efficient disruption of the CCR5 locus. We directly compared activity in primary CD4 T cells using mRNA nuclease delivery. Indel frequency was estimated using both the T7 endonuclease assay and a re-cleavage assay (RCA; where genomic DNA surrounding the cleavage site is amplified and digested in vitro with the original LHE used to create the MTAL). NHEJ rates were 65-82% for the TALEN vs. 44-61% (6 experiments) for the MTAL using these respective assays. Next, we coupled mRNA-mediated nuclease expression with AAV delivery of a donor template. We co-delivered mRNA with a range of doses of an AAV-CCR5-GFP donor (containing 1.3kb CCR5 homology arms and an internal MND-GFP expression cassette). Cell viability was not significantly different; and control experiments using AAV BFP (lacking homology arms) demonstrated that infection and expression rates were equivalent. Strikingly, the proportion of cells with sustained GFP expression (at day 16) was up to 5-fold higher in MTAL treated cells (across a range of AAV doses; n=6, independent donors); with the greatest differences observed when access to donor template was limiting. Molecular analysis verified HDR in both test groups. While the mechanism for these differences remains incompletely defined, preliminary comparisons of HR:NHEJ ratios using single cell analysis suggest that alternative DNA repair pathways may be preferentially utilized by the 3’ vs 5’ overhangs generated by LHE vs Fok1 nucleases, respectively. Taken together, our findings suggest that the MTAL platform and the co-delivery method described here may provide significant clinical utility in future editing applications in human hematopoietic cells.