Allogeneic transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs engraft and reconstitute multiple hematopoietic lineages in vivo and can be engineered to express multiple antibodies simultaneously (in pre-clinical models). Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the foundation for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.

Blood transfusion plays a vital role in modern medicine, but frequent shortages occur. Ex vivo manufacturing of red blood cells (RBCs) from universal donor cells offers a potential solution, yet the high cost of recombinant cytokines remains a barrier. Erythropoietin (EPO) signaling is crucial for RBC development, and EPO is among the most expensive media components. To address this challenge, we develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) using design-build-test cycles and genome editing. By integrating synEPOR at the endogenous EPOR locus in O-negative induced pluripotent stem cells, we achieve equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production using small molecules compared to EPO-supplemented cultures. This approach dramatically reduces culture media costs. Our strategy not only addresses RBC production challenges but also demonstrates how protein and genome engineering can introduce precisely regulated cellular behaviors, potentially improving scalable manufacturing of a wide range of clinically relevant cell types.

Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of synEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media—EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how protein engineering and genome engineering may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated synthetic biology applications.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34+ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a β-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.

Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance. Edited human hematopoietic stem and progenitor cells (HSPCs) maintain multi-lineage repopulation capacity in vivo, and edited primary human T cells potently inhibit infection by both CCR5-tropic and CXCR4-tropic HIV-1. Modification rates facilitated complete loss of CCR5-tropic replication and up to a 2,000-fold decrease in CXCR4-tropic replication without CXCR4 locus disruption. This multi-factor editing strategy in HSPCs could provide a broad approach for autologous HSCT as a functional cure for both CCR5-tropic and CXCR4-tropic HIV-1 infections.

OBJECTIVE:To identify factors that impact parental willingness to consent to research studies conducted for their children during visits to pediatric emergency departments (EDs).METHODS:Parents and guardians of children receiving care in our pediatric ED were approached and asked if they would be willing to let their child participate in a research study requiring the child to complete an electronic questionnaire. No such questionnaire existed, however, because the primary purpose was to ascertain the parent's willingness to let their child participate. All parents were debriefed and informed of the true purpose of the study and asked to complete a survey themselves to help understand factors that influenced their initial decision of whether to consent. Bivariate tests and logistic regression were used to evaluate unadjusted and adjusted associations between parent and patient characteristics and parental consent decision.RESULTS:We approached 431 eligible parents about the hypothetical research study involving their children, and 386 (89.6%) consented for their children to participate. After the debriefing, 392 (91.0%) parents consented to complete the parental survey. We observed statistically significant associations between shorter length of ED stay to approach for consent for the study ( P = 0.048) as well as longer travel time ( P = 0.03) and willingness to consent in bivariate analysis, though this did not hold in regression analysis. Regression analysis revealed parents of children who have previously participated in research had 79 times lower odds of consenting to participate in our study adjusted for parent race, ethnicity, actual and perceived length of stay, travel time to the ED, and altruism.CONCLUSIONS:A high proportion of parents consented to their child participating in research in our ED with previous child participation in research being associated with lower odds of parental consent even when adjusted for other factors. Our findings may inform future research practices and studies investigating parental perceptions and motivations surrounding research studies.

Autologous transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5 -null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs maintain engraftment capacity and multi-lineage potential in vivo and can be engineered to express multiple antibodies simultaneously. Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro . This work lays the groundwork for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis 1 . These lesions are precursors for blood cancers 2 – 6 , but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2 , ASXL1 , SF3B1 and SRSF2 , but this effect was not seen in clones with driver mutations in DNMT3A . TCL1A was not expressed in normal or DNMT3A -mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Genome editing is a powerful tool that enables precise changes in the genetic code of a cell. Genome-edited hematopoietic stem and progenitor cells (HSPCs) yield genome-corrected cells of all lineages, yet the only cell type of clinical relevance in red blood cell (RBC) disorders is the RBC. Most applications of genome editing have sought to correct disease-causing mutations of monogenic diseases. However, human genetic variation reveals variants that confer positive health benefits as well. In this study we took advantage of a naturally occurring human variant to increase erythropoietic output from genome-edited HSPCs. The variant leads to a condition termed congenital erythrocytosis (CE), a rare phenotype in which people have higher than normal levels of RBCs and consequently elevated hemoglobin. It is caused by truncations in the erythropoietin receptor (tEPOR) in which the intracellular inhibitory domain to erythropoietin (EPO) signaling is eliminated. The genetic cause of this phenotype was identified in a family of a Finnish Olympic gold medal-winning cross-country skier who was found to have levels of hemoglobin >50% higher than normal. One of the challenges in gene therapy is to achieve sufficient engraftment of genetically engineered cells to have a clinical effect. Here we sought to give edited cells a selective advantage such that low levels of engraftment might still result in a clinical benefit through use of genome editing to recreate the CE phenotype by engineering tEPOR into HSPCs in different ways. We hypothesized we could recapitulate the CE variant using CRISPR/Cas9 genome editing of HSPCs to create insertions/deletions (indels) at the EPOR locus near the site of the mutation ( EPOR c.1316G>A) and then transfer the cells into culture media that promotes erythroid differentiation (Fig. 1). We demonstrated increased erythropoietic output from cells containing truncating mutations in EPOR, measured by an increase in indel formation throughout erythroid differentiation (1.3-3.8-fold increase). Because not all indels created cause truncations in EPOR, we explored if we could amplify this effect by integrating an AAV6 repair template that introduces a stop codon into EPOR followed by a BGH-polyA tail and GFP marker driven by a constitutive UbC promoter. This strategy enriched for GFP + cells with the truncation even more dramatically as they developed during erythroid differentiation (2.5-4.6-fold increase). We also found a substantial increase in the total number of cells produced from the tEPOR edited condition at the end of differentiation (3.3 times greater fold increase compared to mock edited cells). Importantly, we observed no enrichment in cells kept in HSPC media or cultured in RBC media without EPO, indicating this effect is EPO-dependent (Fig. 2). Next, we investigated whether a tEPOR cDNA could be targeted to the CCR5 safe harbor locus under ubiquitous expression or to the HBA1 RBC-specific safe harbor site imparting high levels of erythroid specific expression. Integration of the tEPOR cDNA at both loci yielded strong enrichment of edited alleles in an EPO-dependent manner when put through erythroid differentiation (6.2-8.6-fold increase at CCR5 and 2.9-5.2 at HBA1). Lastly, we sought to pair the tEPOR cDNA with a clinical edit for β-thalassemia to boost production of disease-corrected RBCs. We showed that through use of bi-cistronic cassettes we were able to drive a >2-fold enrichment of edited alleles over the course of differentiation. Additionally, we were able to pair the tEPOR cDNA and clinical edit for β-thalassemia through multiplexed gene editing at EPOR and HBA1, respectively, and again demonstrated enrichment of edited alleles. Ultimately, we believe this work has the potential to amplify the efficacy of gene and cell therapies for blood disorders currently in the clinic. Expression of tEPOR could be integrated into any treatment for blood disorders that involve transplantation of HSPCs, including both allogeneic and autologous bone marrow transplants. Further, as low chimerism in the bone marrow could produce high levels of peripheral RBCs using our strategy, it has the potential to reduce the morbidity of myeloablation regimens currently required for cure of blood disorders. This work, in effect, has the potential to improve the safety and accessibility to currently available treatments for the millions of patients affected by RBC disorders worldwide.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

Introduction: Oxygen is delivered to all tissues in the body through the hemoglobin molecule uniquely present in red blood cells (RBCs). Proper production and function of RBCs is therefore critical to human health. However, genetic defects of the hematologic system such as anemias and hemoglobin disorders (broadly termed “hemoglobinopathies”) are among the most common genetic disorders in the world. Therefore, developing strategies to regulate RBC development may allow researchers and clinicians to increase therapeutic potential of hematologic cell and gene therapies. Erythropoietin (EPO) signaling is indispensable to RBC development. In its native form, two EPO receptor (EPOR) monomers dimerize in the presence of EPO cytokine to initiate a downstream signaling cascade. Prior work has shown that conformational requirements of EPOR dimerization are rather “loose” and signaling may also be initiated by agonistic diabodies or in the context of chimeric receptors. While these chimeric receptors have limited clinical applicability, inducible dimerization domains such as engineered FKBPs are currently being used in clinical trials in the form of inducible safety switches. Due to the modular nature of cytokine receptors, we hypothesized that inducible dimerization domains (such as the FKBP domain) could be paired with endogenous receptors and signaling pathways to generate chimeric receptors that place RBC development under the control of a bioavailable small molecule - and therefore in the control researchers and clinicians. This study presents a set of chimeric, synthetic receptors that support HSPC output towards the erythroid lineage via a non-immunogenic, non-toxic small molecule (AP30187, hereafter termed “BB”). Methods: In-vitro editing was performed in primary human CD34+ HSPCs by electroporating Cas9 protein complexed with sgRNA along with an AAV6 DNA repair template to facilitate gene insertion via homology-directed repair (HDR). Bulk edited CD34+ HSPCs were then differentiated into RBCs using established differentiation protocols in the presence and absence of both erythropoietin and BB. Differentiation was determined by staining for RBC surface markers (CD71 and GPA) and subsequent flow cytometry analysis. Erythropoietic output was determined by cell density counts. Allele frequency of the edited cell population was determined through droplet digital PCR. Results: We generated seven FKBP-EPOR chimeric receptors - with the basic structure of SFFV-[FKBP-EPOR]-2A-YFP-bGH - that placed the FKBP binding domain at different regions of the endogenous receptor (Figure 1) and analyzed their ability to support RBC differentiation in the absence of EPO but with BB. Out of the seven constructs we found a set of receptors - 1.4 and 1.5 - that demonstrated erythropoietin-independent, BB-mediated erythroid differentiation at 108% and 94% mock RBC differentiation respectively. Construct 1.5 - dubbed inducible EPOR (iEPOR) - was chosen over 1.4 for further optimization because it was also no longer EPO responsive. We further optimized the design of iEPOR by 1) introducing a secretory signal peptide to improve trafficking of the receptor to the cell surface; 2) inclusion of a naturally occurring EPOR truncation that results in supraphysiological RBC differentiation; and 3) modification of the genomic insertion site and promoter of the iEPOR cassette to generate a toolbox of expression profiles. Our results suggest that iEPOR mimics the native EPOR signaling cascade with high fidelity and sensitivity according to bulk RNA-Seq data. We also observe a significant increase in erythropoietic output for iEPOR version 1.4 (6.7-fold by day 11 of RBC differentiation in +BB over -BB conditions, P=0.003), which was further enhanced by the above modifications for vector iEPOR version 3.5.2 (12.9-fold by day 11 of RBC differentiation in +BB over -BB conditions, P<0.0001). Additionally, iEPOR is demonstrated to be erythropoietin-independent, dose-responsive, and tunable via expression from different loci and promoters. Conclusion:iEPORs represent a powerful new tool to alter the cellular dynamics of red blood cell development. The work demonstrated in this study has the potential to dramatically improve the efficacies of existing gene therapies, offer a route to EPO-free ex-vivo RBC production, as well as serve as a new tool by which to better understand stem cell biology and differentiation.

Pathogenic biallelic variants in HSD17B3 result in 17 beta-hydroxysteroid dehydrogenase 3 (17 beta-HSD3) deficiency, variable disruption of testosterone production, and phenotypic diversity among 46, XY individuals with differences of sexual development (DSDs). We performed quad whole exome sequencing (WES) on two male siblings with microphallus, perineal hypospadias, and bifid scrotum and their unaffected parents. Both male siblings were compound heterozygous for a rare pathogenic HSD17B3 variant (c.239 G > A, p.R80Q) previously identified among individuals with 17 beta-HSD3 deficiency and a HSD17B3 variant (c.641A > G, p.E214 G) of uncertain significance. Following WES, the siblings underwent hCG stimulation testing with measurement of testosterone, androstenedione, and dihydrotestosterone which was non-diagnostic. To confirm pathogenicity of the HSD17B3 variants, we performed transient transfection of HEK-293 cells and measured conversion of radiolabeled androstenedione to testosterone. Both HSD17B3 variants decreased conversion of radiolabeled androstenedione to testosterone. As pathogenic HSD17B3 variants are rare causes of 46, XY DSD and hCG stimulation testing may not be diagnostic for 17 beta-HSD3 deficiency, WES in 46, XY individuals with DSDs can increase diagnostic yield and identify genomic variants for functional characterization of disruption of testosterone production.

INTRODUCTION:Niemann-Pick C (NPC) is an autosomal recessive disease due to defective NPC1 or NPC2 proteins resulting in endo-lysosomal storage of unesterified cholesterol in the central nervous system and liver. Acute liver disease in the newborn period may be self-limited or fatal. 2-hydroxypropyl-β-cyclodextrin (2HPBCD) is a cholesterol-binding agent that reduces lysosomal cholesterol storage. We have enrolled 3 infants 0-6 months old with direct hyperbilirubinemia due to NPC1 or NPC2 liver disease in a Phase I/II open label clinical trial of intravenous 2HPBCD. METHODS:Infants received intravenous 2HPBCD twice a week for 6 weeks, followed by monthly infusion for 6-months. Primary outcome measure was reduction of plasma (3β,5α,6β-trihydroxy-cholan-24-oyl) glycine (TCG), a bile acid generated from cholesterol sequestered in lysosome. RESULTS:Three participants completed this protocol. A fourth patient received intravenous 2HPBCD under an emergency investigational new drug study but later expired from her underlying condition. The three protocol patients are living and have improved liver enzymes and TCG. No patient has experienced a drug-related adverse event. CONCLUSION:Intravenous 2HPBCD was tolerated in three infants with liver disease due to NPC.