ALBERT

Description: Current autologous stem cell transplantation and gene therapy strategies are limited by the inability to identify a true hematopoietic stem cell (HSC) propulation that reliably predicts engraftment. The ability to enrich and target a specific HSC pool and predict engraftment levels of the gene-modified cells would be a major advance, reducing manufacturing costs and off-target effects. Here we describe a distinct HSC phenotype, conserved between humans and nonhuman primates (NHP) which overcomes this limitation. We used our NHP stem cell transplantation and gene therapy model to study the engraftment potential of phenotypically distinct hematopoietic stem and progenitor cell (HSPC) subpopulations. We were able to identify an exclusive HSPC subpopulation capable of multi-lineage engraftment (Figure 1A and 1B). This HSPC subpopulation (denoted "I") accounts for ~3-5% of the entire CD34+ cell population in primed bone marrow, reducing the number of cells targeted for cell and gene therapy approaches by 20 to 30-fold. For autologous transplants, only 300-400K HSPCs/kg body weight were required to achieve rapid neutrophil and plateletet recovery within 9-10 and 19-20 days, respectively. Stable 30-35% gene-marking was obeserved in all blood lineages including T cell, B cell, NK cells, granulocyte, monocytes/macrophages, erythrocytes and platelets. Complete reconstitution of the bone marrow compartment was achieved within

Description: Key Points This study is the first to show that genome-editing approaches can modify multilineage, long-term repopulating cells in a large animal model. We demonstrate that the persistence of genome-edited hematopoietic stem cells can be tracked in vivo in a mutation-specific manner.

Description: BACKGROUND A key event in the lifecycle of Human Immunodeficiency Virus (HIV) is permanent integration into the infected cells genome. In addition to allowing long-term persistence of the virus, this results in a trackable mark carried in all infected cells. Active HIV replication represses cellular pathways, preventing further cell division. This would imply that any specific integration site (IS) which is clonally expanded either during active or repressed viral infection arises from either a dormant/inactive virus, or is perturbing local gene expression, leading to increased cell proliferation. Alternatively, a cell carrying HIV provirus could proliferate due to T-cell specific antigen stimulation. By analyzing the patterns of integration sites detected in cell cultures and tissue samples from animal models of HIV infection, we can better understand the basic virology of integration site selection and determine what may potentially drive infected cells to persist despite effective treatment regimens. METHODS Jurkat reporter cell lines or primary human CD4+ cells were cultured and infected with various strains of HIV including both CCR5 and CXCR4 tropic viruses. Infected cells were cultured up to 21 days post infection, then analyzed for HIV proviral integration sites by next-generation sequencing. For in vivo studies, NSG mice were infused with human CD34+ hematopoietic stem/progenitor cells, resulting in a reconstituted human immune system including high levels of CD4+ T cells capable of sustaining HIV infection. After 16 weeks post-challenge, tissues were collected and subjected to integration site analysis for HIV proviral DNA. Identified integration sites were mapped and compared across multiple parameters to identify chromosomal regions and associated genes enriched for integration events, as well as clonally expanded cells in vivo. RESULTS Genome-wide analysis of HIV integration sites reveals a remarkably similar chromosomal landscape both in tissue culture infection of Jurkat cells and in vivo infection data (Figure 1), as well as across multiple HIV strains. As previously observed, the majority of integrations occur near or within gene coding regions thought to be actively transcribed at time of infection. However, certain areas of the genome, and even unique genes, are enriched for IS in individual samples. In addition to these genomic regions of enrichment, we also observe specific clonal outgrowth of unique integration events in genes previously unidentified in the literature. Three genes in particular exhibit a significant increase of integration events during acute infection which are 3x higher than predicted by random chance alone. We also observe integration events in genes that have been documented by other labs in HIV+ clinical patient samples, however in our active infection models, we do not see those specific genes enriched or expanded. This could indicate that these genes play a role in persistence that is only present during anti-retroviral therapy which suppresses active replication. CONCLUSIONS We have cataloged the most extensive HIV IS library to date in both relevant tissue culture models and in vivo infection studies, including over 245,000 unique integration events and three different HIV strains commonly used in research. Genome-wide correlation studies reveal regions significantly enriched for HIV integrations and genes which repeatedly exhibit clonal outgrowth in multiple animals. These types of studies are now being applied to human patient samples to determine if latency and persistence of infection can be mapped to unique integration events or genes of interest. Such information may indicate when and how the latent HIV reservoir is seeded and what types of therapy or treatments are most effective at targeting and eliminating these populations. Circos plot comparing HIV integrations sites (IS) identified either during in vitro cell culture infections (black bars), or in vivo infection studies using humanized mice (red bars). The outer ring is composed of human chromosomes each of which are divided into 25kB fragment bins. Total number of unique integration sites identified in each bin is represented by the height of the histogram bars. The in vitro IS concentric ring scale represents increments of 25 outwards up to 250 while the in vivo IS scales inwards in increments of 2 up to 16. Figure 1 Comparison of in vitro vs in vivo HIV Integration Sites. Figure 1. Comparison of in vitro vs in vivo HIV Integration Sites. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership.

Description: Summary: Several models of hematopoietic reconstitution after transplant have been proposed with multiple methods of tracking individual clones in both animal models and patients. Lentivirus-mediated gene therapy is the most widely applied strategy for marking individual cells, tracked by either provirus integration site analysis (ISA) or by proviral DNA barcode sequencing (DBS). Two previous studies have applied one of each of these methods to track hematopoiesis in a nonhuman primate model of myeloablative autologous transplantation. The report using ISA described long-term, multi-lineage hematopoietic clones emerging at 1 year after transplantation [Kim S. et al., Cell Stem Cell, 2014]. The report using DBS described only early engraftment up to 9 months after transplant, precluding long term analysis [Wu C. et al., Cell Stem Cell, 2014]. We applied a combination of ISA and DBS methods to follow longitudinal hematopoiesis in a total of six nonhuman primates following autologous transplant in the myeloablative setting, tracking 〉120,000 individual clones for up to 10 years. We measured hematopoietic lineage reconstitution, as well as spatial and temporal contributions of individual clones within the bone marrow niche and peripheral blood. Here we applied a stringent definition of the hematopoietic stem cell (HSC) phenotype in vivo (i.e. shared clonal identity between short-lived granulocytes and long-lived B cells with multiple time points of detection), to assess HSC contributions after engraftment. Strikingly, we demonstrate sustained contribution of HSC clones occurs as early as 1 month after transplant and that minor clonal waves continuously emerge when the measureable clonal pool is small in size, but not when the initial engrafting clonal pool is robust. Nearly one half of all HSC clones identified over the length of follow-up were detectable in the periphery within a few months after transplant by both ISA and DBS methods. However, we also identify technical differences between these clone tracking methods that can obscure biological interpretation. This study demonstrates an early and robust phase of multi-lineage hematopoietic reconstitution after myeloablative transplantation in the nonhuman primate, suggesting a single subpopulation of CD34+ cells which maintains clonal stability over the lifetime of the recipient in the autologous setting. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

Description: For more than 30 years, hematopoietic stem cell (HSC) research has been performed according to the classical model of human hematopoiesis suggesting an early segregation of lymphoid and erythro-myeloid potentials. However, several studies have recently proposed a great variety of models for the human blood hierarchy showing alternative lineage relationships and read-outs for multipotent HSCs. While the debate about hematopoietic lineage relationships is still ongoing, consequences of these findings and the challenges they pose for the development of treatment strategies for hematological diseases and malignancies are rarely discussed. A critical factor for the development of stem cell therapies is the availability of a reliable and robust read-out for multipotent HSCs/MPPs (multipotent progenitor cells) supporting the development of all blood cell lineages. Unfortunately, the current gold standard, NOD/SCID mouse xenograft repopulation assay does not support erythrocyte and megakaryocyte development and, thus, does not fully read-out multi-lineage potential of human HSCs/MPP. In addition, development of therapeutic approaches in the mouse model is not possible due to differences in cell surface marker expression, physiology, life span, and the demand on stem cell self-renewal and differentiation compared to humans. The pigtail macaque (PM; Macaca nemestrina) and the rhesus macaque (RM: Macaca mulatta) share a close evolutionary relationship with humans and have been used as a pre-clinical model system to study basic HSC biology or to develop specific HSC gene therapy approaches. Surprisingly, however, a comparison of hematopoietic subpopulations and the hierarchical organization of defined lineages has not been performed between NHPs and humans. This will be a critical factor for a better understanding of the newly defined blood lineage associations and hierarchies, as well as the development of treatment approaches based on these lineages. Here, we comprehensively analyzed all known markers of human hematopoiesis in the NHP to identify subpopulations of candidate NHP hematopoietic stem and progenitor cells (HSPCs) and then validated HSPC phenotypes of these fractions with functional in vitro read outs. We further evaluated and compared lineage relationships between these subpopulations to recently proposed models of human hematopoiesis to determine whether conservation of hematopoiesis exists with the goal of informing studies evaluating treatments for hematological diseases in the NHP model. We show for the first time a phenotypic mapping strategy in NHP hematopoietic cells predicting a revised model of hematopoiesis. Similar to humans, NHP HSCs give rise to multipotent progenitors (MPPs), followed by a segregation of lympho-myeloid, erythro-myeloid, and megakaryocytic lineages (see figure). Conservation of hematopoietic lineage relationships was confirmed by RNA expression analysis of corresponding subpopulations. In summary, we identified corresponding human and NHP hematopoietic subpopulations, which share phenotypical, functional and transcriptional properties in both species, validating the NHP as an excellent pre-clinical model system for HSC biology and the development of novel HSC-based treatment approaches. Figure Figure. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.