Intracellular RNase activity dampens zinc finger nuclease-mediated gene editing in hematopoietic stem and progenitor cells.

Over the past decade, numerous gene-editing platforms which alter host DNA in a highly specific and targeted fashion have been described. Two notable examples are zinc finger nucleases (ZFNs), the first gene-editing platform to be tested in clinical trials, and more recently, CRISPR/Cas9. Although CRISPR/Cas9 approaches have become arguably the most popular platform in the field, the therapeutic advantages and disadvantages of each strategy are only beginning to emerge. We have established a nonhuman primate (NHP) model that serves as a strong predictor of successful gene therapy and gene-editing approaches in humans; our recent work shows that ZFN-edited hematopoietic stem and progenitor cells (HSPCs) engraft at lower levels than CRISPR/Cas9-edited cells. Here, we investigate the mechanisms underlying this difference. We show that optimized culture conditions, including defined serum-free media, augment engraftment of gene-edited NHP HSPCs in a mouse xenograft model. Furthermore, we identify intracellular RNases as major barriers for mRNA-encoded nucleases relative to preformed enzymatically active CRISPR/Cas9 ribonucleoprotein (RNP) complexes. We conclude that CRISPR/Cas9 RNP gene editing is more stable and efficient than ZFN mRNA-based delivery and identify co-delivered RNase inhibitors as a strategy to enhance the expression of gene-editing proteins from mRNA intermediates.

Leaf mass area determines water use efficiency through its influence on carbon gain in rice mutants.

Saving water and enhancing rice productivity are consensually the most important research goals globally. While increasing canopy cover would enhance growth rates by higher photosynthetic carbon gain, an accompanied increase in transpiration would have a negative impact on saving water as well as for sustainability under water-limited conditions. Increased water use efficiency (WUE) by virtue of higher carbon assimilatory capacity can significantly circumvent this trade-off. Here, we report leaf mass area (LMA) has an important canopy architecture trait which when combined with superior carboxylation efficiency (CE) would achieve higher water productivity in rice. A set of 130 ethyl methanesulfonate induced mutants of an upland cultivar Nagina-22 (N22), was screened for leaf morphological traits leading to the identification of mutants differing in LMA. The wild-type, N22, along with a selected low-LMA (380-4-3) and two high-LMA mutants (392-9-1 and 457-1-3), all with comparable total leaf area, were raised under well-watered (100% Field Capacity (FC)) and water-limited (60% FC) conditions. Low Δ13 C and a higher RuBisCO content in high-LMA mutants indicated higher carboxylation efficiency, leading to increased carbon gain. Single parent backcross populations developed by crossing high and the low-LMA mutants with N22, separately, were screened for LMA, Δ13 C and growth traits. Comparison of dry matter accumulation per unit leaf area among the progenies differing in LMA and Δ13 C reiterated the association of LMA with CE. Results illustrated that high-LMA when combined with higher CE (low Δ13 C) lead to increased WUE and growth rates.

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA-, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

Allele-specific analysis of single parent backcross population identifies HOX10 transcription factor as a candidate gene regulating rice root growth.

Understanding the molecular and physiological mechanisms of trait diversity is crucial for crop improvement to achieve drought adaptation. Root traits such as high biomass and/or deep rootedness are undoubtedly important drought adaptive traits. The major aim of this investigation was to functionally characterize a set of ethyl methane sulfonate-induced rice mutants for root traits. We report the identification of a high-root biomass mutant through a novel screening strategy for yield and Δ13 C measurements. The high-root mutant (392-9-1) thus identified, had a 66% higher root biomass compared to wild-type (Nagina-22). Better maintenance of leaf turgor and carbon assimilation rates resulted in lower drought susceptibility index in 392-9-1. Targeted resequencing revealed three non-synonymous single nucleotide variations in 392-9-1 for the genes HOX10, CITRATE SYNTHASE and ZEAXANTHIN EPOXIDASE. Segregation pattern of phenotype and mutant alleles in a single parent backcross F2 population revealed a typical 3:1 segregation for each of the mutant alleles. The number of F2 progeny with root biomass equal to or greater than that of 392-9-1 represented approximately one-third of the population indicating a major role played by HOX10 gene in regulating root growth in rice. Allele-specific Sanger sequencing in contrasting F2 progenies confirmed the co-segregation of HOX10 allele with the root biomass. The non-synonymous mutations in the other two genes did not reveal any specific pattern of co-segregation with root phenotype, indicating a strong role of HOX10, an upstream transcription factor, in regulating root biomass in rice.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation.

Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.

Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy.

Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

Correction: Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS.

[This corrects the article DOI: 10.1371/journal.ppat.1006753.].

Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS.

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.

Ipilimumab-induced necrotic myelopathy in a patient with metastatic melanoma: A case report and review of literature.

Ipilimumab is a novel humanized monoclonal antibody directed against cytotoxic T lymphocyte antigen 4, a T-cell surface molecule involved in down-regulation and suppression of the T cell response to stimuli. Patients treated with ipilimumab are at risk for immune-related adverse events involving the skin, digestive tract, liver and endocrine organs. Few case reports of immune-related adverse effects involving central or peripheral nervous system due to ipilimumab are published. These include inflammatory myopathy, aseptic meningitis, severe meningo-radiculo-neuritis, temporal arteritis, Guillain-Barre syndrome, and posterior reversible encephalopathy syndrome. We report the first case of ipilimumab-induced progressive necrotic myelopathy.

Development and characterization of microsatellite markers for Morus spp. and assessment of their transferability to other closely related species.

BACKGROUND: Adoption of genomics based breeding has emerged as a promising approach for achieving comprehensive crop improvement. Such an approach is more relevant in the case of perennial species like mulberry. However, unavailability of genomic resources of co-dominant marker systems has been the major constraint for adopting molecular breeding to achieve genetic enhancement of Mulberry. The goal of this study was to develop and characterize a large number of locus specific genic and genomic SSR markers which can be effectively used for molecular characterization of mulberry species/genotypes. RESULT: We analyzed a total of 3485 DNA sequences including genomic and expressed sequences (ESTs) of mulberry (Morus alba L.) genome. We identified 358 sequences to develop appropriate microsatellite primer pairs representing 222 genomic and 136 EST regions. Primers amplifying locus specific regions of Dudia white (a genotype of Morus alba L), were identified and 137 genomic and 51 genic SSR markers were standardized. A two pronged strategy was adopted to assess the applicability of these SSR markers using mulberry species and genotypes along with a few closely related species belonging to the family Moraceae viz., Ficus, Fig and Jackfruit. While 100% of these markers amplified specific loci on the mulberry genome, 79% were transferable to other related species indicating the robustness of these markers and the potential they hold in analyzing the molecular and genetic diversity among mulberry germplasm as well as other related species. The inherent ability of these markers in detecting heterozygosity combined with a high average polymorphic information content (PIC) of 0.559 ranging between 0.076 and 0.943 clearly demonstrates their potential as genomic resources in diversity analysis. The dissimilarity coefficient determined based on Neighbor joining method, revealed that the markers were successful in segregating the mulberry species, genotypes and other related species into distinct clusters. CONCLUSION: We report a total of 188 genomic and genic SSR markers in Morus alba L. A large proportion of these markers (164) were polymorphic both among mulberry species and genotypes. A substantial number of these markers (149) were also transferable to other related species like Ficus, Fig and Jackfruit. The extent of polymorphism revealed and the ability to detect heterozygosity among the cross pollinated mulberry species and genotypes render these markers an invaluable genomic resource that can be utilized in assessing molecular diversity as well as in QTL mapping and subsequently mulberry crop improvement through MAS.

Cell interactions and patterned intercalations shape and link epithelial tubes in C. elegans.

Many animal organs are composed largely or entirely of polarized epithelial tubes, and the formation of complex organ systems, such as the digestive or vascular systems, requires that separate tubes link with a common polarity. The Caenorhabditis elegans digestive tract consists primarily of three interconnected tubes-the pharynx, valve, and intestine-and provides a simple model for understanding the cellular and molecular mechanisms used to form and connect epithelial tubes. Here, we use live imaging and 3D reconstructions of developing cells to examine tube formation. The three tubes develop from a pharynx/valve primordium and a separate intestine primordium. Cells in the pharynx/valve primordium polarize and become wedge-shaped, transforming the primordium into a cylindrical cyst centered on the future lumenal axis. For continuity of the digestive tract, valve cells must have the same, radial axis of apicobasal polarity as adjacent intestinal cells. We show that intestinal cells contribute to valve cell polarity by restricting the distribution of a polarizing cue, laminin. After developing apicobasal polarity, many pharyngeal and valve cells appear to explore their neighborhoods through lateral, actin-rich lamellipodia. For a subset of cells, these lamellipodia precede more extensive intercalations that create the valve. Formation of the valve tube begins when two valve cells become embedded at the left-right boundary of the intestinal primordium. Other valve cells organize symmetrically around these two cells, and wrap partially or completely around the orthogonal, lumenal axis, thus extruding a small valve tube from the larger cyst. We show that the transcription factors DIE-1 and EGL-43/EVI1 regulate cell intercalations and cell fates during valve formation, and that the Notch pathway is required to establish the proper boundary between the pharyngeal and valve tubes.

Laminin is required to orient epithelial polarity in the C. elegans pharynx.

The development of many animal organs involves a mesenchymal to epithelial transition, in which cells develop and coordinate polarity through largely unknown mechanisms. The C. elegans pharynx, which is an epithelial tube in which cells polarize around a central lumen, provides a simple system with which to understand the coordination of epithelial polarity. We show that cell fate regulators cause pharyngeal precursor cells to group into a bilaterally symmetric, rectangular array of cells called the double plate. The double plate cells polarize with apical localization of the PAR-3 protein complex, then undergo apical constriction to form a cylindrical cyst. We show that laminin, but not other basement membrane components, orients the polarity of the double plate cells. Our results provide in vivo evidence that laminin has an early role in cell polarity that can be distinguished from its later role in basement membrane integrity.