Genome editing in large animal models Maynard, Lucy H.; Humbert, Olivier; Peterson, Christopher W. ...
Molecular therapy,
11/2021, Letnik:
29, Številka:
11
Journal Article
Recenzirano
Odprti dostop
Although genome editing technologies have the potential to revolutionize the way we treat human diseases, barriers to successful clinical implementation remain. Increasingly, preclinical large animal ...models are being used to overcome these barriers. In particular, the immunogenicity and long-term safety of novel gene editing therapeutics must be evaluated rigorously. However, short-lived small animal models, such as mice and rats, cannot address secondary pathologies that may arise years after a gene editing treatment. Likewise, immunodeficient mouse models by definition lack the ability to quantify the host immune response to a novel transgene or gene-edited locus. Large animal models, including dogs, pigs, and non-human primates (NHPs), bear greater resemblance to human anatomy, immunology, and lifespan and can be studied over longer timescales with clinical dosing regimens that are more relevant to humans. These models allow for larger scale and repeated blood and tissue sampling, enabling greater depth of study and focus on rare cellular subsets. Here, we review current progress in the development and evaluation of novel genome editing therapies in large animal models, focusing on applications in human immunodeficiency virus 1 (HIV-1) infection, cancer, and genetic diseases including hemoglobinopathies, Duchenne muscular dystrophy (DMD), hypercholesterolemia, and inherited retinal diseases.
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In this review, Maynard and colleagues outline how large animal models are being applied to optimize state-of-the-art gene editing therapies for the clinic. Specific focus is given to large animal models of HIV-1, cancer, and various heritable genetic diseases.
Risk factors for grades 2-4 acute graft-versus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients ...of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD.
Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) have produced impressive minimal residual disease–negative (MRD-negative) complete remission (CR) rates in ...patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the factors associated with durable remissions after CAR T-cell therapy have not been fully elucidated. We studied patients with relapsed/refractory B-ALL enrolled in a phase 1/2 clinical trial evaluating lymphodepletion chemotherapy followed by CD19 CAR T-cell therapy at our institution. Forty-five (85%) of 53 patients who received CD19 CAR T-cell therapy and were evaluable for response achieved MRD-negative CR by high-resolution flow cytometry. With a median follow-up of 30.9 months, event-free survival (EFS) and overall survival (OS) were significantly better in the patients who achieved MRD-negative CR compared with those who did not (median EFS, 7.6 vs 0.8 months; P < .0001; median OS, 20.0 vs 5.0 months; P = .014). In patients who achieved MRD-negative CR by flow cytometry, absence of the index malignant clone by IGH deep sequencing was associated with better EFS (P = .034). Stepwise multivariable modeling in patients achieving MRD-negative CR showed that lower prelymphodepletion lactate dehydrogenase concentration (hazard ratio HR, 1.38 per 100 U/L increment increase), higher prelymphodepletion platelet count (HR, 0.74 per 50 000/μL increment increase), incorporation of fludarabine into the lymphodepletion regimen (HR, 0.25), and allogeneic hematopoietic cell transplantation (HCT) after CAR T-cell therapy (HR, 0.39) were associated with better EFS. These data allow identification of patients at higher risk of relapse after CAR T-cell immunotherapy who might benefit from consolidation strategies such as allogeneic HCT. This trial was registered at www.clinicaltrials.gov as #NCT01865617.
•Baseline platelet count, lactate dehydrogenase, and lymphodepletion regimen impact EFS in patients in MRD-negative CR after CD19 CAR T-cell.•Allogeneic HCT after CD19 CAR T-cell therapy is well tolerated and may improve EFS.
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Recently in Nature, Gupta et al. (2019) reported that a second patient has achieved HIV-1 remission/functional cure after allogeneic hematopoietic stem cell transplantation from a donor carrying a ...mutation in the CCR5 coreceptor. We highlight both patients’ treatments and summarize efforts to develop a broader, more patient-friendly approach.
Recently in Nature, Gupta et al. (2019) reported that a second patient has achieved HIV-1 remission/functional cure after allogeneic hematopoietic stem cell transplantation from a donor carrying a mutation in the CCR5 coreceptor. We highlight both patients’ treatments and summarize efforts to develop a broader, more patient-friendly approach.
...using the pre-clinical nonhuman primate (NHP) stem cell transplantation and gene therapy model, sort-purified CD34+CD90+CD45RA− cells reduced the target cell number up to 20-fold, were responsible ...for rapid short- and persistent long-term multilineage engraftment of gene-modified cells, and accurately predicted recovery onset.3 Despite obvious differences in the methodology and target cell phenotype, all strategies successfully reduced the number of target cells without compromising the efficiency of gene modification or engraftment potential. ...a similar selection approach is currently being studied in the allogeneic setting in severe-combined immunodeficiency patients (ClinicalTrials.gov: NCT02963064). To increase availability, a promising option is the use of CPF-independent, semi- or fully-automated and portable closed-system applications, such as the CliniMACS Prodigy (Miltenyi Biotech), to perform a bead-based purification of phenotypically defined HSPCs.4 Complementary to this system, the availability of a closed-system cell sorter, such as the MACSQuant Tyto (Miltenyi Biotech), has the potential to turn flow-based cell-sorting for HSC gene therapy into a portable and CPF-independent approach that could potentially reach more than 50 million patients worldwide. ...we think the future is very bright for HSC gene therapy and editing.
•Correction of the sickle-cell mutation and disease phenotypes is achieved by in vivo HSC transduction with vectorized prime editors.•Our approach for in vivo HSC prime editing that does not require ...HSC transplantation and myeloablation should simplify HSC gene therapy.
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Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the β-globin gene. Current gene therapy studies are mainly focused on lentiviral vector–mediated gene addition or CRISPR/Cas9–mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor–expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of βS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.
Li and colleagues report on a novel gene-therapy approach to sickle cell disease. Rather than ex vivo manipulation with lentiviral gene addition or CRISPR/Cas9-mediated fetal hemoglobin reactivation, the authors describe injection of a nonintegrating prime editor–expressing vector into a sickle mouse model with correction of over 40% of hemoglobin S alleles in vivo. Though several features need to be optimized, this technique offers a potential for gene-therapy delivery in resource-poor settings.
Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human ...embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.
Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are ...persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.
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