Alloengraftment without significant toxicity or GVHD in CD45 antibody-drug conjugate-conditioned Fanconi anemia mice.

ABSTRACT: Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage-Sca1+cKit+ cells within 48 hours. Fanca-/- recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc-/- and Fancg-/- recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca-/- recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.

Enforced gut homing of murine regulatory T cells reduces early graft-versus-host disease severity.

Damage to the gastrointestinal tract following allogeneic hematopoietic stem cell transplantation is a significant contributor to the severity and perpetuation of graft-versus-host disease. In preclinical models and clinical trials, we showed that infusing high numbers of regulatory T cells reduces graft-versus-host disease incidence. Despite no change in in vitro suppressive function, transfer of ex vivo expanded regulatory T cells transduced to overexpress G protein-coupled receptor 15 or C-C motif chemokine receptor 9, specific homing receptors for colon or small intestine, respectively, lessened graft-versus-host disease severity in mice. Increased regulatory T cell frequency and retention within the gastrointestinal tissues of mice that received gut homing T cells correlated with lower inflammation and gut damage early post-transplant, decreased graft-versus-host disease severity, and prolonged survival compared with those receiving control transduced regulatory T cells. These data provide evidence that enforced targeting of ex vivo expanded regulatory T cells to the gastrointestinal tract diminishes gut injury and is associated with decreased graft-versus-host disease severity.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

Human CD19-specific switchable CAR T-cells are efficacious as constitutively active CAR T-cells but cause less morbidity in a mouse model of human CD19+ malignancy.

Current Food and Drug Administration (FDA)-approved CD19-specific chimeric antigen receptor (CAR) T-cell therapies for B-cell malignancies are constitutively active and while efficacious, can cause morbidity and mortality. Their toxicities might be reduced if CAR T-cell activity was regulatable rather than constitutive. To test this, we compared the efficacies and morbidities of constitutively active (conventional) and regulatable (switchable) CAR (sCAR) T-cells specific for human CD19 (huCD19) in an immune-competent huCD19+ transgenic mouse model.Conventional CAR (CAR19) and sCAR T-cells were generated by retrovirally transducing C57BL/6 (B6) congenic T-cells with constructs encoding antibody-derived single chain Fv (sFv) fragments specific for huCD19 or a peptide neoepitope (PNE), respectively. Transduced T-cells were adoptively transferred into huCD19 transgenic hemizygous (huCD19Tg/0 ) B6 mice; healthy B-cells in these mice expressed huCD19Tg Prior to transfer, recipients were treated with a lymphodepleting dose of cyclophosphamide to enhance T-cell engraftment. In tumor therapy experiments, CAR19 or sCAR T-cells were adoptively transferred into huCD19Tg/0 mice bearing a syngeneic B-cell lymphoma engineered to express huCD19. To regulate sCAR T cell function, a switch protein was generated that contained the sCAR-specific PNE genetically fused to an anti-huCD19 Fab fragment. Recipients of sCAR T-cells were injected with the switch to link sCAR effector with huCD19+ target cells. Mice were monitored for survival, tumor burden (where appropriate), morbidity (as measured by weight loss and clinical scores), and peripheral blood lymphocyte frequency.CAR19 and sCAR T-cells functioned comparably regarding in vivo expansion and B-cell depletion. However, sCAR T-cells were better tolerated as evidenced by the recipients' enhanced survival, reduced weight loss, and improved clinical scores. Discontinuing switch administration allowed healthy B-cell frequencies to return to pretreatment levels.In our mouse model, sCAR T-cells killed huCD19+ healthy and malignant B-cells and were better tolerated than CAR19 cells. Our data suggest sCAR might be clinically superior to the current FDA-approved therapies for B-cell lymphomas due to the reduced acute and chronic morbidities and mortality, lower incidence and severity of side effects, and B-cell reconstitution on cessation of switch administration.

Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.

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.

Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti-human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.

Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.

Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing.

Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4+ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4+ T cell and up to 88% of CD34+ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4+ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion.

BACKGROUND AIMS: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg. RESULTS: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. DISCUSSION: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile-desirable properties that support the possibility of off-the-shelf Treg therapeutics.

Repurposing a novel anti-cancer RXR agonist to attenuate murine acute GVHD and maintain graft-versus-leukemia responses.

The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.

Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs.

Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T→G•C adenine base editor (ABE) to correct two different COL7A1 mutations in primary fibroblasts derived from RDEB patients. ABE enabled higher COL7A1 correction efficiencies than previously reported HDR efforts. Moreover, ABE obviated the need for a repair template, and minimal indels or editing at off-target sites was detected. Base editing restored the endogenous type VII collagen expression and function in vitro. We also treated induced pluripotent stem cells (iPSCs) derived from RDEB fibroblasts with ABE. The edited iPSCs were differentiated into mesenchymal stromal cells, a cell population with therapeutic potential for RDEB. In a mouse teratoma model, the skin derived from ABE-treated iPSCs showed the proper deposition of C7 at the dermal-epidermal junction in vivo. These demonstrate that base editing provides an efficient and precise genome editing method for autologous cell engineering for RDEB.

Phenotype categorization of neurofibromatosis type I and correlation to NF1 mutation types.

Neurofibromatosis type 1 (NF1) is caused by heterozygous mutation in the NF1 gene. NF1 is one of the most common human genetic diseases. However, the overall genotype-phenotype correlation has not been known, due to a wide spectrum of genotypic and phenotypic heterogeneity. Here we describe the detailed clinical and genetic features of 427 Korean NF1 patients from 389 unrelated families. Long range PCR and sequencing of genomic DNA with multiplex ligation-dependent probe amplification analysis identified 250 different NF1 mutations in 363 families (93%), including 94 novel mutations. With an emphasis on phenotypes requiring medical attention (classified and termed: NF1+), we investigated the correlation of NF1+ and mutation types. NF1+ was more prevalent in patients with truncating/splicing mutations and large deletions than in those with missense mutations (59.6%, 64.3% vs. 36.6%, p = 0.001). This difference was especially significant in the patients younger than age 19 years. The number of items in NF1+ was a higher in the former groups (0.95 ± 0.06, 1.18 ± 0.20 vs. 0.56 ± 0.10, p = 0.002). These results suggest that mutation types are associated not only with higher prevalence of severe phenotypes in NF1 but also with their earlier onset.

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.

The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.

Donor and host B7-H4 expression negatively regulates acute graft-versus-host disease lethality.

B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4-/- versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4-/- recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4-/- versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4-/- donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4-/- recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4-/- mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.

Human CD19-Targeted Mouse T Cells Induce B Cell Aplasia and Toxicity in Human CD19 Transgenic Mice.

The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have ∼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.

CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression.

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34⁺ hematopoietic stem cells and peripheral blood T-cells. CD34⁺ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

Inside out: regenerative medicine for recessive dystrophic epidermolysis bullosa.

Epidermolysis bullosa is classified as a genodermatosis, an inherited genetic skin disorder that results in severe, chronic skin blistering with painful and life-threatening complications. Although there is currently no cure for epidermolysis bullosa, concurrent advances in gene and stem cell therapies are converging toward combinatorial therapies that hold the promise of clinically meaningful and lifelong improvement. Recent studies using hematopoietic stem cells and mesenchymal stromal/stem cells to treat epidermolysis bullosa have demonstrated the potential for sustained, effective management of the most severe cases. Furthermore, advances in the use of gene therapy and gene-editing techniques, coupled with the development of induced pluripotent stem cells from patients with epidermolysis bullosa, allow for autologous therapies derived from a renewable population of cells that are patient-specific. Here we describe emerging treatments for epidermolysis bullosa and other genodermatoses, along with a discussion of their benefits and limitations as effective therapies.

Incorporation of Immune Checkpoint Blockade into Chimeric Antigen Receptor T Cells (CAR-Ts): Combination or Built-In CAR-T.

Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade.