Jaya Sahni

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.

SLC41A1 Mg 2+ transport is regulated via Mg 2+ -dependent endosomal recycling through its N-terminal cytoplasmic domain

SLC41A1 (solute carrier family 41, member A1) is a recently described vertebrate member of the MgtE family of Mg(2+) transporters. Although MgtE transporters are found in both prokaryotic and eukaryotic organisms, and are highly conserved, little is known about the regulation of their Mg(2+) transport function. In the present study, we have shown that endogenous SLC41A1 transporter expression is post-transcriptionally regulated by extracellular Mg(2+) in TRPM7 (transient receptor potential cation channel, subfamily M, member 7)-deficient cells, suggesting that SLC41A1 transporters underlie a novel plasma membrane Mg(2+) transport function. Consistent with this conclusion, structure-function analyses of heterologous SLC41A1 transporter expression demonstrate that SLC41A1 transporters exhibit the same plasma membrane orientation as homologous bacterial MgtE proteins, are capable of complementing growth of TRPM7-deficient cells only when the Mg(2+) transporting pore is intact, and require an N-terminal cytoplasmic domain for Mg(2+)-dependent regulation of lysosomal degradation and surface expression. Taken together, our results indicate that SLC41A1 proteins are a central component of vertebrate Mg(2+) transport systems, and that their Mg(2+) transport function is regulated primarily through an endosomal recycling mechanism involving the SLC41A1 N-terminal cytoplasmic domain.

The SLC41 family of MgtE-like magnesium transporters.

Magnesium is one of the most predominant intracellular divalent cations and is requisite to the regulation of a diverse array of cellular functions. Although accumulating data from multiple studies have begun to illuminate the critical role(s) played by Mg(2+) transporters in pathways involved in cell signaling, metabolism, growth and proliferation, there is still a lack of understanding of the underlying molecular mechanisms that govern those various functions. In this review, we focus on the recently described SLC41 family of magnesium transporters, two members of which have been shown to mediate Mg(2+) uptake and transport, and highlight what is known about their expression, localization, and function, as well as their roles and contributions to cellular Mg(2+) transport.

High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k “Helper” Proteins

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

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.

pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A) Sequences.

Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s).

The B. subtilis MgtE Magnesium Transporter Can Functionally Compensate TRPM7-Deficiency in Vertebrate B-Cells

Recent studies have shown that the vertebrate magnesium transporters Solute carrier family 41, members 1 and 2 (SLC41A1, SLC41A2) and Magnesium transporter subtype 1 (MagT1) can endow vertebrate B-cells lacking the ion-channel kinase Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) with a capacity to grow and proliferate. SLC41A1 and SLC41A2 display distant homology to the prokaryotic family of Mg2+ transporters, MgtE, first characterized in Bacillus subtilis. These sequence similarities prompted us to investigate whether MgtE could potentially compensate for the lack of TRPM7 in the vertebrate TRPM7-deficient DT40 B-cell model system. Here, we report that overexpression of MgtE is able to rescue the growth of TRPM7-KO DT40 B-cells. However, contrary to a previous report that describes regulation of MgtE channel gating by Mg2+ in a bacterial spheroplast model system, whole cell patch clamp analysis revealed no detectable current development in TRPM7-deficient cells expressing MgtE. In addition, we observed that MgtE expression is strongly downregulated at high magnesium concentrations, similar to what has been described for its vertebrate homolog, SLC41A1. We also show that the N-terminal cytoplasmic domain of MgtE is required for normal MgtE channel function, functionally confirming the predicted importance of this domain in regulation of MgtE-mediated Mg2+ entry. Overall, our findings show that consistent with its proposed function, Mg2+ uptake mediated by MgtE is able to restore cell growth and proliferation of TRPM7-deficient cells and supports the concept of functional homology between MgtE and its vertebrate homologs.

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.

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.

567. Enhanced Efficiency of CRISPR-Mediated Gene Knock-Out in Primary Human T-Cells

RNA-guided endonuclease (RGEN) technology has great promise for enabling efficient editing of essentially any target genomic locus. Here, we have evaluated application of S. pyogenes Cas9 for gene editing in primary human T-cells using mRNA-mediated delivery of a first generation spCas9 and adeno-associated virus (AAV) to drive guide RNA expression. We find that spCas9-mediated editing using this approach can achieve targeted gene disruption rates at the TCRa locus of up to 30%. Furthermore, evaluation of the dose response of editing efficiency at different Cas9 mRNA doses and over a range of AAV MOI suggests that editing efficiency is limited primarily by AAV-driven guide RNA expression. Evaluation of several approaches to achieve higher editing efficiencies led to the development of a novel method capable of achieving up to 90% TCRa disruption at reduced AAV MOI in selected cell populations. These results suggest that a Cas9-mRNA/AAV-guide approach can be applied to effectively disrupt multiple individual genes in primary human T-cells, and highlight an innovate method through which CRISPR/Cas9 technology may be applied with enhanced efficiency.