non-viral-engineering-primary-human-t-cells-via-homology-mediated-end

Non-Viral Engineering of Primary Human T Cells <em>via</em> Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) ...

University of Minnesota

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease characterized by mutations to the α-L-iduronidase (IDUA) gene resulting in inactivation of the IDUA enzyme. The loss of IDUA protein results in the progressive accumulation of glycosaminoglycans within the lysosomes resulting in severe, multi-organ system pathology. Gene replacement strategies have relied on the use of viral or nonviral gene delivery systems. Drawbacks to these include laborious production procedures, poor efficacy due to plasmid-borne gene silencing, and the risk of insertional mutagenesis. This report demonstrates the efficacy of a nonintegrating, minicircle (MC) DNA vector that is resistant to epigenetic gene silencing in vivo. To achieve sustained expression of the immunogenic IDUA protein we investigated the use of a tissue-specific promoter in conjunction with microRNA target sequences. The inclusion of microRNA target sequences resulted in a slight improvement in long-term expression compared to their absence. However, immune modulation by costimulatory blockade was required and permitted for IDUA expression in MPS I mice that resulted in the biochemical correction of pathology in all of the organs analyzed. MC gene delivery combined with costimulatory pathway blockade maximizes safety, efficacy, and sustained gene expression and is a new approach in the treatment of lysosomal storage disease.

Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of α-L-iduronidase in mice with mucopolysaccharidosis type I.

Engineered zinc finger nucleases (ZFNs) are a tool for genome manipulation that are of great interest to scientists in many fields. To meet the needs of researchers wishing to employ ZFNs, an inexpensive, rapid assembly procedure would be beneficial to laboratories that do not have access to the proprietary reagents often required for ZFN production. Using freely available sequence data derived from the Zinc Finger Targeter database, we developed a protocol for synthesis and directed insertion of user-defined ZFNs into a versatile plasmid expression system. This oligonucleotide-based isothermal DNA assembly protocol was used to determine whether we could generate functional nucleases capable of endogenous gene editing. We targeted the human α-l-iduronidase (IDUA) gene on chromosome 4, mutations of which result in the severe lysosomal storage disease mucopolysaccharidosis type I. In approximately 1 week we were able to design, assemble, and test six IDUA-specific ZFNs. In a single-stranded annealing assay five of the six candidates we tested performed at a level comparable to or surpassing previously reported ZFNs. One of the five subsequently showed nuclease activity at the endogenous genomic IDUA locus. To our knowledge, this is the first demonstration of in silico-designed, oligonucleotide-assembled, synthetic ZFNs, requiring no specialized templates or reagents that are capable of endogenous human gene target site activity. This method, termed CoDA-syn (context-dependent assembly-synthetic), should facilitate a more widespread use of ZFNs in the research community.

Synthetic zinc finger nuclease design and rapid assembly.

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10 kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.

TALEN-based gene correction for epidermolysis bullosa.

Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder. 

Fanconi anemia gene editing by the CRISPR/Cas9 system.

Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies. 

Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases.

The ability to rationally target disease-causing mutations has been made possible with programmable nucleases with the CRISPR/Cas9 system representing a facile platform for individualized gene-based medicine. In this study we employed footprint free reprogramming of fibroblasts from a patient with mutations to the Fanconi anemia I (FANCI) gene to generate induced pluripotent stem cells (iPSC). This process was accomplished without gene complementation and the resultant iPSC were able to be gene corrected in a robust manner using the Cas9 nickase. The self-renewing iPSC that were maintained under feeder free conditions were differentiated into cells with characteristics of definitive hematopoiesis. This defined and highly efficient procedure employed small molecule modulation of the hematopoietic differentiation pathway and a vascular induction technique to generate hematopoietic progenitors. In sum, our results demonstrate the ability to induce patient derived FA cells to pluripotency for patient specific therapeutic cell derivation.

CRISPR/Cas9 Targeted Gene Editing and Cellular Engineering in Fanconi Anemia.

Mucopolysaccharidosis type I (MPS IH) is a lysosomal storage disease (LSD) caused by inactivating mutations to the alpha-L-iduronidase (IDUA) gene. Treatment focuses on IDUA enzyme replacement and currently employed methods can be non-uniform in their efficacy particularly for the cardiac and craniofacial pathology. Therefore, we undertook efforts to better define the pathological cascade accounting for treatment refractory manifestations and demonstrate a role for the renin angiotensin system (RAS) using the IDUA mouse model. Perturbation of the RAS in the aorta was more profound in male animals suggesting a causative role in the observed gender dimorphism and angiotensin receptor blockade (ARB) resulted in improved cardiac function. Further, we show the ability of losartan to prevent shortening of the snout, a common craniofacial anomaly in IDUA mice. These data show a key role for the RAS in MPS associated pathology and support the inclusion of losartan as an augmentation to current therapies.

Angiotensin receptor blockade mediated amelioration of mucopolysaccharidosis type I cardiac and craniofacial pathology.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the  gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing  mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation  in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB.

CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa.

Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 () primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct a  gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase).  function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary  cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells.

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

Over the past decade, immunotherapeutic strategies-mainly targeting the PD-1-PD-L1 immune checkpoint axis-have altered cancer treatment for many solid tumours, but few patients with gastrointestinal forms of cancer have benefited to date. There remains an urgent need to extend immunotherapy efficacy to more patients while addressing resistance to current immune checkpoint inhibitors. The aim of this study was to determine the safety and anti-tumour activity of knockout of CISH, which encodes cytokine-inducible SH2-containing protein, a novel intracellular immune checkpoint target and a founding member of the SOCS family of E3-ligases, using tumour infiltrating lymphocyte (TILs) genetically edited with CRISPR-Cas9 in patients with metastatic gastrointestinal epithelial cancers.

Targeting the intracellular immune checkpoint CISH with CRISPR-Cas9-edited T cells in patients with metastatic colorectal cancer: a first-in-human, single-centre, phase 1 trial.

Article	Year
Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of α-L-iduronidase in mice with mucopolysaccharidosis type I. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 21081900	2011
Synthetic zinc finger nuclease design and rapid assembly. Human gene therapy\| PubMed ID: 21663559	2011
TALEN-based gene correction for epidermolysis bullosa. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 23546300	2013
Fanconi anemia gene editing by the CRISPR/Cas9 system. Human gene therapy\| PubMed ID: 25545896	2015
Evaluation of TCR Gene Editing Achieved by TALENs, CRISPR/Cas9, and megaTAL Nucleases. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 26502778	2016
CRISPR/Cas9 Targeted Gene Editing and Cellular Engineering in Fanconi Anemia. Stem cells and development\| PubMed ID: 27538887	2016
Angiotensin receptor blockade mediated amelioration of mucopolysaccharidosis type I cardiac and craniofacial pathology. Journal of inherited metabolic disease\| PubMed ID: 27743312	2017
CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa. NPJ Regenerative medicine\| PubMed ID: 28250968	2016
CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells. International journal of molecular sciences\| PubMed ID: 28613254	2017
Correction of Fanconi Anemia Mutations Using Digital Genome Engineering. International journal of molecular sciences\| PubMed ID: 35955545	2022
Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair. Nature biomedical engineering\| PubMed ID: 38092857	2024
Targeting the intracellular immune checkpoint CISH with CRISPR-Cas9-edited T cells in patients with metastatic colorectal cancer: a first-in-human, single-centre, phase 1 trial. The Lancet. Oncology\| PubMed ID: 40315882	2025

Anthony P. DeFeo

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