Abstract CAR T cell activity in solid tumors is limited by off-tumor toxicity and functional suppression from the tumor microenvironment (TME). To address these challenges, we have developed AB-1015, an autologous ICT cell product intended for use in ovarian cancer. AB-1015 incorporates two functional modules: an” AND” logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition, and a dual shRNA-miR targeting FAS and PTPN2 to resist TME suppression and to improve ICT cell function. The AB-1015 logic gate consists of a priming receptor (PrimeR) against ALPG/P and an inducible anti-MSLN CAR that is upregulated upon PrimeR engagement. At basal state, only a small percentage of AB-1015 cells have detectable CAR present at low MFI. The percentage of AB-1015 cells that express CAR, as well as the CAR MFI, increase in response to increasing levels of ALPG. This unique feature of the logic gate aims to increase the tumor specificity of AB-1015, given that ALPG/P and MSLN are not co-expressed in the normal tissues. To functionally test the dual-antigen specificity, AB-1015 cells were co-cultured with K562 cells engineered with only one target antigen (ALPG or MSLN), both antigens (ALPG and MSLN), or neither antigen. AB-1015 demonstrated potent killing of K562-ALPG/MSLN cells, and minimal activity against K562 cells expressing only MSLN. In addition, AB-1015 showed over 100-fold increase in IFNg production when co-cultured with K562-ALPG/MSLN cells, compared to co-culture with K562-MSLN cells that do not express ALPG. The dual-antigen specificity of the logic gate was further assessed in vivo using a dual flank tumor xenograft model where one tumor expressed both ALPG and MSLN, and the contralateral tumor expressed MSLN alone. Compared with the RNP group, the constitutive anti-MSLN CAR demonstrated tumor reduction on both flanks. In contrast, the activity of AB-1015 was specific to the ALPG+MSLN+ tumor. The anti-tumor activity of AB-1015 was established in an intraperitoneal OVCAR3 ovarian xenograft model that resembles high-grade serous ovarian cancer histology. AB-1015 demonstrated potent anti-tumor activity as demonstrated by a decrease in bioluminescent signal from the tumors treated with AB-1015. To further increase the stringency of our preclinical models, we engineered the subcutaneous MSTO xenograft model to express FASL. In this model, conventional anti-MSLN CAR T cells failed to control tumor outgrowth. In contrast, AB-1015 resists FASL suppression via knockdown of FAS on the ICT surface. As a result, AB-1015 is capable of completely clearing these otherwise difficult-to-treat tumors in this model. In summary, maximal AB-1015 effector function depends on PrimeR engagement with ALPG/P and CAR binding to MSLN. AB-1015 demonstrates superior potency compared with conventional anti-MSLN CAR T cells and is resistant to ovarian TME suppression in preclinical studies. Based on these promising preclinical data, AB-1015 is being studied in a phase I clinical trial (NCT05617755) for patients with platinum-resistant ovarian cancer. Citation Format: Jun Feng, Jasper Williams, Hongruo Yun, Dina Polyak, James Zhang, Michelle Nguyen, Irene Scarfo, Jessica Fuhriman, Aaron Cooper, Jennifer McDevitt, Stephen Santoro. AB-1015, an Integrated Circuit T (ICT) cell therapy containing an ALPG/MSLN logic gate and FAS/PTPN2 shRNA-miR, for the treatment of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B080.

Supplementary Figures 1-4 from BMI-1 Promotes Ewing Sarcoma Tumorigenicity Independent of CDKN2A Repression

Supplementary Data from Intercohort Gene Expression Co-Analysis Reveals Chemokine Receptors as Prognostic Indicators in Ewing's Sarcoma

Abstract Clinically effective adoptive T cell therapy for the treatment of solid tumors will require robust T cell expansion, persistence, and potency. The Janus-kinase signal transducer and activator of transcription (JAK-STAT) pathway governs T cell activation and differentiation, and can thereby serve as a critical regulator of these properties. To take advantage of the benefits of STAT signaling in programming an antitumor T-cell response, we used synthetic biology to create a library of proteins, termed Synthetic Pathway Activators (SPAs) which constitutively drive STAT signaling without the need for external cytokine input. SPAs can be designed to engage activity of multiple STAT family transcription factors at variable levels through rational design. We have developed several classes of SPAs, including but not limited to Class I SPAs, which primarily increase pSTAT3 activity, and Class II SPAs, which increase pSTAT5 activity. When constitutively expressed in ArsenalBio Integrated Circuit T (ICT) cells, SPAs result in significant enhancements in T-cell potency and expansion. Repetitive stimulation assays, wherein T cells are challenged with tumor cells every 2 days, reveal that Class I SPAs result in 6-log or higher improved tumor cell clearance over a 2-week assay period. Across various mouse xenograft models, SPA-expressing ICTs reach at least 6-fold improved tumor growth inhibition. RNAseq and ATACseq analysis indicate dramatic changes to gene expression profiles in T cells expressing Class I SPAs, with maintenance of T cell stem-like phenotypes, and restricted accessibility of various exhaustion marker genes. Importantly, despite significantly increased levels of expansion, ICTs equipped with SPAs are not immortalized, showing no signs of cytokine-independent outgrowth. In addition, SPA-expressing ICT cells rapidly contract following tumor clearance in-vivo. The SPA platform represents a novel, tunable, and T cell intrinsic approach for engineering cell fates that result in potent anti-tumor properties. Citation Format: Thomas J. Gardner, Beatriz Millare, Anzhi Yao, Ashley Cass, Suchismita Mohanty, Jeremy Chen, Alma Gomez, David DeTomaso, Manching Ku, Lionel Berthoin, Meng Lim, Azalea Ong, Vince Thomas, Nicholas Quant, Brian Hsu, Amy-Jo Casbon, Natalie Bezman, Aaron Cooper, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Tunable STAT activation by synthetic pathway activators (SPAs) increases engineered T-cell potency and persistence. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4073.

Supplementary Table 1 from BMI-1 Promotes Ewing Sarcoma Tumorigenicity Independent of CDKN2A Repression

Chimeric antigen receptor T cell (CAR T) therapy has demonstrated unprecedented therapeutic activity in hematologic malignancies. However, generating potent clinical responses against solid tumors remains a challenge for CAR T therapy. As the field strives to improve the therapeutic efficacy of CAR T cells with novel target antigens and enhanced potency, the risks of on-target toxicity pose a major barrier to progress. To address these challenges, we have developed engineered CAR T cells to target solid tumors through AND logic gates, where CAR expression is conditionally induced by a transcription factor released from a priming receptor (PrimeRTM) upon binding to the PrimeR antigen. The AND gate limits off-tumor toxicity as it requires both CAR and PrimeR antigen expression in the tumor microenvironment. To ensure PrimeR expression and signal transduction upon antigen binding, while minimizing residual ‘‘leaky’’ CAR induction in the absence of PrimeR antigen, we screened hundreds of PrimeR binders using both arrayed and pooled strategies. In an arrayed strategy, we engineered T cells from four donors in multiwell plates using CRISPR-mediated, non-viral, site-specific integration of logic gates bearing a variable PrimeR binder and a fixed MSLN CAR. In addition, we employed a pooled screening strategy, where we engineered T cells from two independent donors with a pool containing a subset of >300 of the same logic gates. Engineered T cells from both strategies were co-cultured with cell lines bearing either both CAR and PrimeR antigens or a single antigen, in order to evaluate fidelity and on-target functionality. In the arrayed setting, on-target functionality was quantified based on the levels of CAR induction, cytokine secretion, T cell activation, and target cell killing in the presence of both antigens, while fidelity was assessed based on the absence of these activity signals in the presence of a single antigen. In the pooled setting, sorting based on functional markers was performed and sequencing was used to quantify the relative abundance of cells with each logic gate in different sorted populations. On-target activity and circuit fidelity were then quantified based on enrichments in different sorted populations. Results from the pooled and arrayed screens were highly concordant. We combined the screen readouts to nominate a small set of PrimeR binders that exhibited both high fidelity and on-target functionality. We confirmed the desired characteristics of these binders with targeted arrayed screens in additional conditions as well as in in-vivo models. We have applied both screen strategies to select a small set of leads from hundreds of candidate PrimeR binders in the context of a logic-gated MSLN CAR. As pooled and arrayed screens come with different sets of limitations and advantages, both serve as important tools for the effective selection of receptors in the development of novel cell therapies. Citation Format: Li Wang, Sofia Kyriazopoulou Panagiotopoulou, Rona Harari-Steinfeld, Dasmanthie De Silva, Michelle Tan, Laura Lim, Angela Boroughs, Cate Sue, Jon Chen, Jamie Thomas, Mary Chua, Ed Yashin, Christine Shieh, Ryan Fong, Sophie Xu, Grace Zheng, Brendan Galvin, Aaron Cooper, Tarjei Mikkelsen, Nicholas Haining. High throughput screening strategies in the development of logic gated cell therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5329.

Abstract CAR T cell therapies for solid tumors are limited by a paucity of tumor-specific single target antigens and insufficient potency due to limitations of CAR T biology. Logic gated (LG) CARs increase control of cell therapy activation and target cell killing by requiring recognition of two or more antigens through AND or AND-NOT Boolean logic. They represent an attractive strategy for cell therapy to improve the therapeutic index by increasing specificity for tumor antigens. We created an AND logic gate cassette deliverable by CRISPR-mediated, non-viral, site-specific integration into human T cells. This logic gate activates expression of a MSLN-targeting CAR upon ALPP/G binding of a priming receptor (PrimeR) within high-grade serous carcinoma ovarian tumors. The PrimeR triggers proteolytic release of a chimeric, fully human transcription factor, which then translocates to the nucleus to induce expression of a minigene encoding a CAR. The PrimeR is built only from human protein sequences, reducing theoretical risk of cell therapy immunogenicity and rejection within a patient. In order to test the specificity of our LG CARs, we integrated LG cassettes into a defined site in the T cell genome, and then co-cultured T cells with K562 cells engineered with only one target antigen (K562-ALPG or K562-MSLN), both target antigens (K562-ALPG/MSLN), or neither target antigen (K562). LG CAR T cells only killed K562-ALPG/MSLN cells. The specificity of this LG was also demonstrated in vivo in a dual-flank K562 model, in which only the ALPG/MSLN tumor was inhibited by LG CAR T cells. CAR expression was not observed on LG CAR T cells recovered outside the ALPG/MSLN tumor, and no growth inhibition was observed in K562-MSLN tumors, unlike a constitutive CAR T control. To model priming antigen heterogeneity expected in tumors, we mixed K562-MSLN cells with varying proportions of K562-ALPG/MSLN cells. We found that the presence of only 5-15% ALPG/MSLN cells was sufficient to effect killing of all target cells by the induced MSLN CAR. The ALPG/MSLN LG CAR will be tested as a component of AB-1015, a novel therapy for indications including ovarian, fallopian tube, or primary peritoneal cancer. Citation Format: Jasper Williams, Kevin Martinez, Angela Boroughs, Laura Lim, Marian Sandoval, Cate Sue, Matthew Drever, Anzhi Yao, Joseph Choe, Maxim Sidorov, Sophia Phillips, Dina Polyak, Suchismita Mohanty, Stephen Santoro, Aaron Cooper, W. Nicholas Haining. Logic gates controlled by priming receptors increase specificity and potency of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2854.

Background Generating potent clinical responses against solid tumors remains a challenge for CAR T cell therapy. This lack of efficacy is likely due in part to reduced on-tumor activity in the tumor microenvironment and the lack of appropriate target antigens that are expressed on tumor cells but not on critical healthy tissues. We addressed the second of these challenges by engineering T cells to target tumors only upon recognition of two antigens through AND Boolean logic. Methods In our efforts to develop logic gated cell therapies we generated hundreds of binders for the priming receptor (PrimeR) and the CAR receptor. Here we demonstrate two strategies used for functional screening of PrimeR binders to identify binders with desired sensitivity and fidelity in driving the specific on-target expression of a fixed CAR. In an arrayed strategy, we engineered T cells from 4 donors in multiwell plates using CRISPR-mediated, non-viral, site-specific integration of circuits bearing ~1000 PrimeR binders and receptor architectures with a fixed MSLN CAR. In addition, we employed a pooled screening strategy in which we engineered 2 donors of T-cells with a pool containing a subset of >300 unique PrimeR binders with a fixed MSLN CAR, using a similar engineering protocol. Engineered T cells from both strategies were co-cultured with target cell lines to evaluate targeting fidelity and on-target functionality. In the arrayed setting, functional readouts (activation markers, cytokine secretion) were measured and reported directly. In the pooled setting, sorting based on functional markers was performed at end-point and sequencing was used to determine the enrichment of specific binders. Circuit fidelity was assessed by the lack of CAR expression and/or T-cell activation in response to target cell lines expressing the cytolytic antigen alone, or neither target antigens. On-target functionality was assessed by quantifying secretion of key cytokines in response to dual antigen stimulation. Results We combined these metrics to filter out PrimeRs that allowed for CAR expression in the absence of logic gate activation, and to rank the remaining binders by their ability to drive CAR expression in the presence of both antigens. To account for multiple criteria when ranking binders, we used desirability functions, scaling each measurement to a (0–1) range, and using geometric means to combine desirabilities across different criteria. Conclusions As each strategy comes with a different set of limitations and advantages, both serve as important tools for the effective selection of binders and receptors in the development of novel cell therapies.

Background Chimeric antigen receptor (CAR) T cell therapy has emerged as an important new tool in the treatment of cancers. However, the complexity of the enhancements used is limited by the amount of genetic information that can be integrated into the genome. Our approach utilizes Integrated Circuit T (ICT) cells, which are engineered to include a large DNA cassette that includes: receptor strategies to target multiple tumor antigens; transcriptional modifications that alter cell state; engineered cytokines and chemokines and variations in the CAR binding and signaling domains. Our first ICT clinical program, AB-1015, is an autologous cell product for the treatment of ovarian cancer. The AB-1015 transgene cassette consists of a logic gate directed against ALPG/P and MSLN and an shRNA-miR module targeting FAS and PTPN2 that enhance potency and confer resistance to the tumor microenvironment. This transgene is delivered into primary T cells via non-viral, site-specific editing into a safe-harbor locus via CRISPR integration of transgenes by electroporation (CITE). CITE has many advantages over viral and other non-viral random integration methods, including more predictable transgene expression and function, reduced risk of unsafe insertional mutagenesis, and efficient integration of large cassettes. Methods To identify candidate genomic loci for CITE-directed gene insertion we used epigenetic analysis, transcriptional profiling, and high-throughput gene-editing of primary T cells. Loci were further characterized using T cell functional assays. Knock-in efficiency and transgene expression stability in primary human T cells were evaluated for all loci. Lead candidate loci were tested for compatibility with complex T cell programs embodied by our integrated circuits, containing a priming receptor (PrimeR, ALPG/P) that triggers the expression of a CAR (MSLN) in response to a priming antigen. The top insertion site, GS94, was further characterized using in silico and empirical approaches. Results GS94 was identified as an optimal locus for CITE-directed gene insertion based upon: 1) stable and high PrimeR expression; 2) high and inducible CAR expression; and 3) a superior T cell cytotoxic and cytokine secretion profile. We were unable to identify any off-target events generated by CITE at GS94, including off-target editing, knock-in and translocations, using a suite of molecular assays including iGUIDE, rhAMPseq, deep whole genome sequencing, and anchored-PCR. Conclusions CITE editing at GS94 is specific and generates highly functional ICT cells. This novel approach to engineering tumor-specific T cells enables the generation of exceptional clinical candidates that both target new cancer types and improve efficacy.

T cell therapies for cancer have shown striking clinical efficacy in different tumor settings. Integration of a transgene cassette expressing an antigen receptor into the genome enables T cells to recognize specific tumor antigens. Viral vectors and transposons can introduce transgenes into primary human T cells. However these approaches cannot control genomic location and have limited control over the copy number of cassette insertions. Here, we used epigenetic analysis, transcriptional profiling, high-throughput gene-editing and T cell functional experiments to identify the optimal genomic locus amenable to non-viral genome targeting. First we analyzed the epigenetic landscape of primary human T cells to identify regions (“gene deserts”) that are accessible but devoid of annotated regulatory elements or coding sequences. Candidate loci were validated using CRISPR/Cas9 genome editing to directly cut and a marker gene cassette was inserted into these sites. Knock-in efficiency and transgene expression stability in primary human T cells were evaluated for all loci. Evaluation of 40 loci demonstrated substantial variability (10 fold) in transgene expression and confirmed that not all sites were equivalent in their ability to sustain transgene expression. Next, we evaluated 8 most promising candidate loci for compatibility with complex T cell programs embodied by integrated circuits, containing a priming receptor (PrimeR) that triggers expression of a CAR in response to a priming antigen. We identified one integration site (termed “GS94”) that supported: 1) stable and high PrimeR expression; 2) high and inducible CAR expression; and 3) a superior T cell cytotoxic and cytokine secretion profile. Finally, we assessed off-target cutting mediated by the CRISPR sgRNA targeting GS94. The sgRNA is the most specific out of the candidates evaluated with iGUIDE and targeted PCR. We were unable to detect any measurable off-target activity by targeted PCR on predicted sites. The GS94 site is a component of the integrated circuit T cell (ICT) therapy, AB-1015, which is now under development for the treatment of high-grade serous carcinoma ovarian tumors. AB-1015 includes an inserted cassette that contains a logic gate and two shRNAs that enhance the potency and solid tumor microenvironment resistance of the AB-1015 ICT cells. These computational and experimental approaches can be generalized to identify new safe harbor sites for different cell types, expanding the genome engineering toolkit for diverse cell therapy applications. Citation Format: Grace X.Y. Zheng, Somya Khare, Brendan Galvin, Robby Moot, Aaron Cooper, Michelle Nguyen, Michelle Tan, Shan Sabri, Audre May, Jun Fung, Anzhi Yao, Andrea Liu, Matt Drever, Steve Santoro, W Nicholas Haining, Tarjei Mikkelsen. Identification of a safe harbor CRISPR-Cas9 integration site for improved cell therapy safety and potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2840.

BackgroundIn solid tumors, CAR T cell efficacy is limited by off-tumor toxicity and suppression by the tumor microenvironment (TME). AB-X is an integrated circuit T cell (ICT cell) intended for the treatment of ovarian cancer. AB-X includes a transgene cassette with two functional modules: 1) an ”AND” logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition; 2) a dual shRNA-miR to resist TME suppression and improve ICT cell function. The AB-X logic gate consists of a priming receptor that induces expression of an anti-mesothelin (MSLN) CAR upon engagement of a ALPG/P (alkaline phosphatase germ-line/placental). The dual shRNA-miR mediates downregulation of FAS and PTPN2. The AB-X DNA cassette is inserted into the T cell genome at a defined novel genomic site via CRISPR-based gene editing.MethodsDual-antigen specificity of the logic gate was assessed in mice harboring MSLN+ and ALPG/P+MSLN+ K562 tumors established on contralateral flanks. Potency was measured in a subcutaneous MSTO xenograft model. Logic-gated ICT cells were compared with MSLN CAR T cells in both models. In vitro, expansion of ICT cells with the FAS/PTPN2 shRNA-miR was evaluated in a 14 day repetitive stimulation assay (RSA). In vivo, expansion and potency were measured in the MSTO xenograft model. An in vitro FAS cross-linking assay was conducted to assess the impact of FAS knockdown on FAS-mediated apoptosis.ResultsLogic-gated ICT cells demonstrated specific activity against ALPG/P+MSLN+ tumors, but had no effect against MSLN+ tumors in the K562 in vivo specificity model. In addition, logic-gated ICT cells demonstrated greater in vivo potency than MSLN CAR T cells in the MSTO xenograft model. In our RSA, ICT cells containing the FAS/PTPN2 shRNA-miR had 8-fold greater expansion than the MSLN CAR T cells. Enhanced expansion was confirmed in vivo with ICT cells demonstrating >10-fold expansion in tumors and peripheral blood, enabling comparable growth inhibition in MSTO xenografts at less than one quarter the dose of the MSLN CAR T cells. Importantly, PTPN2 knockdown resulted in balanced expansion of all T cell subsets, including CD45RA+, CCR7+ memory cells. Lastly, ICT cells containing the FAS/PTPN2 shRNA-miR were resistant to FAS-mediated apoptosis.ConclusionsAB-X ICT cells specifically recognize ALPG/P+MSLN+ tumors, demonstrate superior potency, expansion, and persistence compared with MSLN CAR T cells, and are resistant to ovarian TME suppression. AB-X will be evaluated in clinical trials for treatment of platinum resistant/refractory ovarian cancer.AcknowledgementsWe would like to acknowledge all of our colleagues at Arsenal Biosciences, without whom this work would not have been possible.

BACKGROUND AIMS:Gene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials.METHODS:Data are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/βAS3-FB vector and cryopreserving CD34+ cells from human bone marrow (BM) at clinical scale. In vitro and in vivo characterization of the FCP was performed, showing that all the release criteria were successfully met. In vivo toxicology studies were conducted to evaluate potential toxicity of the Lenti/βAS3-FB LV in the context of a murine BM transplant.RESULTS:Primary and secondary transplantation did not reveal any toxicity from the lentiviral vector. Additionally, vector integration site analysis of murine and human BM cells did not show any clonal skewing caused by insertion of the Lenti/βAS3-FB vector in cells from primary and secondary transplanted mice.CONCLUSIONS:We present here a complete protocol, thoroughly optimized to manufacture, characterize and establish safety of a FCP for gene therapy of SCD.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

We report follow-up of subjects treated in a Phase II study of gene therapy for ADA-SCID. Between 2009 and 2012, ten ADA-deficient SCID patients were treated by γ-retroviral-mediated gene transfer (MND-ADA) to their bone marrow CD34+ cells. The subjects were given non-myeloablative chemotherapy (busulfan @ 90 mg/m2) and were withdrawn from PEG-ADA enzyme replacement therapy (ERT) prior to infusion of autologous gene-modified cells. Subject age at the time of treatment ranged from 3 months to 15 years (median = 11.5 months). Follow-up times range from 2 to 5 years. All but one subject, who was 15-years old at the time of treatment, remain off PEG-ADA ERT with immune reconstitution that reached maximal level between 6 and 12 months after transplant and was maintained thereafter. Vector marking in peripheral blood cells remained consistently detectable (> 0.1 copy/PBMC and ≥ 0.003 copy/granulocyte) at 2 years and later after transplant in subjects who discontinued ERT. These subjects also had PBMC ADA enzymatic activity in the normal range and red blood cell deoxynucleotide levels below 10%. Three subjects have discontinued intravenous immunoglobulin; five subjects have discontinued prophylactic antibiotics. All subjects have polyclonal gene marking with no sign of lymphoproliferative disease. The subjects remain in good health without infections or other complications.

Gene transfer into autologous hematopoietic stem cells by.-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1 alpha promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T-and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 x 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.

Introduction: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known.Methods: In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions.Results: We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status.Conclusion: For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.