Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb.

Coevolutionary interactions are responsible for much of the Earth's biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes' importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

α1B/D-adrenoceptors regulate chemokine receptor-mediated leukocyte migration via formation of heteromeric receptor complexes.

It is known that catecholamines regulate innate immune functions. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family in recombinant systems and that such heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Ligand binding to α1-ARs inhibited migration toward agonists of the CR heteromerization partners of α1B/D-ARs with high potency and 50 to 77% efficacy but did not affect migration induced by a noninteracting CR. Incomplete siRNA knockdown of α1B/D-ARs in THP-1 cells partially inhibited migration toward agonists of their CR heteromerization partners. Complete α1B-AR knockout via CRISPR-Cas9 gene editing in THP-1 cells (THP-1_ADRA1BKO) resulted in 82% reduction of α1D-AR expression and did not affect CR expression. Migration of THP-1_ADRA1BKO cells toward agonists of CR heteromerization partners of α1B/D-ARs was reduced by 82 to 95%. Our findings indicate that CR:α1B/D-AR heteromers are essential for normal function of CR heteromerization partners, provide a mechanism underlying neuroendocrine control of leukocyte trafficking, and offer opportunities to modulate leukocyte and/or cancer cell trafficking in disease processes.

Bi-allelic variants in OGDHL cause a neurodevelopmental spectrum disease featuring epilepsy, hearing loss, visual impairment, and ataxia.

The 2-oxoglutarate dehydrogenase-like (OGDHL) protein is a rate-limiting enzyme in the Krebs cycle that plays a pivotal role in mitochondrial metabolism. OGDHL expression is restricted mainly to the brain in humans. Here, we report nine individuals from eight unrelated families carrying bi-allelic variants in OGDHL with a range of neurological and neurodevelopmental phenotypes including epilepsy, hearing loss, visual impairment, gait ataxia, microcephaly, and hypoplastic corpus callosum. The variants include three homozygous missense variants (p.Pro852Ala, p.Arg244Trp, and p.Arg299Gly), three compound heterozygous single-nucleotide variants (p.Arg673Gln/p.Val488Val, p.Phe734Ser/p.Ala327Val, and p.Trp220Cys/p.Asp491Val), one homozygous frameshift variant (p.Cys553Leufs∗16), and one homozygous stop-gain variant (p.Arg440Ter). To support the pathogenicity of the variants, we developed a novel CRISPR-Cas9-mediated tissue-specific knockout with cDNA rescue system for dOgdh, the Drosophila ortholog of human OGDHL. Pan-neuronal knockout of dOgdh led to developmental lethality as well as defects in Krebs cycle metabolism, which was fully rescued by expression of wild-type dOgdh. Studies using the Drosophila system indicate that p.Arg673Gln, p.Phe734Ser, and p.Arg299Gly are severe loss-of-function alleles, leading to developmental lethality, whereas p.Pro852Ala, p.Ala327Val, p.Trp220Cys, p.Asp491Val, and p.Arg244Trp are hypomorphic alleles, causing behavioral defects. Transcript analysis from fibroblasts obtained from the individual carrying the synonymous variant (c.1464T>C [p.Val488Val]) in family 2 showed that the synonymous variant affects splicing of exon 11 in OGDHL. Human neuronal cells with OGDHL knockout exhibited defects in mitochondrial respiration, indicating the essential role of OGDHL in mitochondrial metabolism in humans. Together, our data establish that the bi-allelic variants in OGDHL are pathogenic, leading to a Mendelian neurodevelopmental disease in humans.

Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

CZON-cutter - a CRISPR-Cas9 system for multiplexed organelle imaging in a simple unicellular alga.

The unicellular alga Cyanidioschyzon merolae has a simple cellular structure; each cell has one nucleus, one mitochondrion, one chloroplast and one peroxisome. This simplicity offers unique advantages for investigating organellar proliferation and the cell cycle. Here, we describe CZON-cutter, an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease for targeted editing of any locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants using fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows us to validate phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. This article has an associated First Person interview with the first author of the paper.

Modular genetic control of social status in a cichlid fish.

Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fish Astatotilapia burtoni is a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. Dominant A. burtoni males possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status in A. burtoni is in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication, A. burtoni possess two androgen receptor (AR) paralogs, ARα and ARß, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutant A. burtoni and performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find that ARß, but not ARα, is required for testes growth and bright coloration, while ARα, but not ARß, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status in A. burtoni is inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity in A. burtoni and other species whose social status relies on social experience.

Knockout of ovary serine protease Leads to Ovary Deformation and Female Sterility in the Asian Corn Borer, Ostrinia furnacalis.

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, a maternally required protease defining embryonic dorsoventral polarity in Drosophila. In this study, we used CRISPR/Cas9-mediated mutagenesis to functionally characterize Osp in the Asian corn borer, Ostrinia furnacalis, a devastating maize pest throughout Asia and Australia. Building on previous knowledge, we hypothesized that knockout of Osp would disrupt embryonic development in O. furnacalis females. To examine this overarching hypothesis, we (1) cloned and characterized Osp from O. furnacalis, (2) designed target sites on exons 1 and 4 to construct a CRISPR/Cas9 mutagenesis system, and (3) documented phenotypic impacts among O. furnacalis Osp mutants. As a result, we (1) examined the temporal-spatial expression profiles of OfOsp, which has an open reading frame of 5648 bp in length and encodes a protein of 1873 amino acids; (2) established O. furnacalis Osp mutants; and (3) documented recessive, female-specific sterility among OfOspF mutants, including absent or deformed oviducts and reduced fertility in female but not male mutants. Overall, the combined results support our initial hypothesis that Osp is required for embryonic development, specifically ovarian maturation, in O. furnacalis females. Given its substantial impacts on female sterility, Osp provides a potential target for the Sterile Insect Technique (SIT) to manage Lepidoptera pests in general and the species complex Ostrinia in particular.

Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction?

Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.

Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and ß, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

Genome-Wide Association Study Identifies a Plant-Height-Associated Gene OsPG3 in a Population of Commercial Rice Varieties.

Plant height is one of the most crucial components of plant structure. However, due to its complexity, the genetic architecture of rice plant height has not been fully elucidated. In this study, we performed a genome-wide association study (GWAS) to determine rice plant height using 178 commercial rice varieties and identified 37 loci associated with rice plant height (LAPH). Among these loci, in LAPH2, we identified a polygalacturonase gene, OsPG3, which was genetically and functionally associated with rice plant height. The rice plant exhibits a super dwarf phenotype when the knockout of the OsPG3 gene occurs via CRISPR-Cas9 gene-editing technology. RNA-Seq analysis indicated that OsPG3 modulates the expression of genes involved in phytohormone metabolism and cell-wall-biosynthesis pathways. Our findings suggest that OsPG3 plays a vital role in controlling rice plant height by regulating cell wall biosynthesis. Given that rice architecture is one of the most critical phenotypes in rice breeding, OsPG3 has potential in rice's molecular design breeding toward an ideal plant height.

CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function.

Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.

Identification of a Rice Leaf Width Gene Narrow Leaf 22 (NAL22) through Genome-Wide Association Study and Gene Editing Technology.

Rice leaf width (RLW) is a crucial determinant of photosynthetic area. Despite the discovery of several genes controlling RLW, the underlying genetic architecture remains unclear. In order to better understand RLW, this study conducted a genome-wide association study (GWAS) on 351 accessions from the rice diversity population II (RDP-II). The results revealed 12 loci associated with leaf width (LALW). In LALW4, we identified one gene, Narrow Leaf 22 (NAL22), whose polymorphisms and expression levels were associated with RLW variation. Knocking out this gene in Zhonghua11, using CRISPR/Cas9 gene editing technology, resulted in a short and narrow leaf phenotype. However, seed width remained unchanged. Additionally, we discovered that the vein width and expression levels of genes associated with cell division were suppressed in nal22 mutants. Gibberellin (GA) was also found to negatively regulate NAL22 expression and impact RLW. In summary, we dissected the genetic architecture of RLW and identified a gene, NAL22, which provides new loci for further RLW studies and a target gene for leaf shape design in modern rice breeding.

Carotenoid biosynthesis is associated with low-temperature adaptation in Rhodosporidium kratochvilovae.

BACKGROUND: Low temperatures greatly limit the growth of microorganisms. Low-temperature adaptation in microorganisms involves multiple mechanisms. Carotenoids are naturally occurring lipid-soluble pigments that act as antioxidants and protect cells and tissues from the harmful effects of free radicals and singlet oxygen. However, studies on the regulation of carotenoid biosynthesis at low temperatures in microorganisms are limited. In this study, we investigated the correlation between carotenoids and low-temperature adaptation in the cold-adapted strain of Rhodosporidium kratochvilovae YM25235. RESULTS: Carotenoid biosynthesis in YM25235 was inhibited by knocking out the bifunctional lycopene cyclase/phytoene synthase gene (RKCrtYB) using the established CRISPR/Cas9 gene-editing system based on endogenous U6 promoters. The carotenoids were extracted with acetone, and the content and composition of the carotenoids were analyzed by spectrophotometry and HPLC. Then, the levels of reactive oxygen species (ROS) and the growth rate in YM25235 were determined at a low temperature. The results indicated that the carotenoid biosynthesis and ROS levels were increased in the YM25235 strain at a low temperature and inhibition of carotenoid biosynthesis was associated with higher ROS levels and a significant decrease in the growth rate of YM25235 at a low temperature. CONCLUSIONS: The regulation of carotenoid biosynthesis was associated with low-temperature adaptation in YM25235. Our findings provided a strong foundation for conducting further studies on the mechanism by which YM25235 can adapt to low-temperature stress.

Extracellular matrix dysfunction in Sorsby patient-derived retinal pigment epithelium.

Sorsby Fundus Dystrophy (SFD) is a rare form of macular degeneration that is clinically similar to age-related macular degeneration (AMD), and a histologic hallmark of SFD is a thick layer of extracellular deposits beneath the retinal pigment epithelium (RPE). Previous studies of SFD patient-induced pluripotent stem cell (iPSC) derived RPE differ as to whether these cultures recapitulate this key clinical feature by forming increased drusenoid deposits. The primary purpose of this study is to examine whether SFD patient-derived iPSC-RPE form basal deposits similar to what is found in affected family member SFD globes and to determine whether SFD iPSC RPE may be more oxidatively stressed. We performed a careful comparison of iPSC RPE from three control individuals, multiple iPSC clones from two SFD patients' iPSC RPE, and post-mortem eyes of affected SFD family members. We also examined the effect of CRISPR-Cas9 gene correction of the S204C TIMP3 mutation on RPE phenotype. Finally, targeted metabolomics with liquid chromatography and mass spectrometry analysis and stable isotope-labeled metabolite analysis were performed to determine whether SFD RPE are more oxidatively stressed. We found that SFD iPSC-RPE formed significantly more sub-RPE deposits (∼6-90 µm in height) compared to control RPE at 8 weeks. These deposits were similar in composition to the thick layer of sub-RPE deposits found in SFD family member globes by immunofluorescence staining and TEM imaging. S204C TIMP3 correction by CRISPR-Cas9 gene editing in SFD iPSC RPE cells resulted in significantly reduced basal laminar and sub-RPE calcium deposits. We detected a ∼18-fold increase in TIMP3 accumulation in the extracellular matrix (ECM) of SFD RPE, and targeted metabolomics showed that intracellular 4-hydroxyproline, a major breakdown product of collagen, is significantly elevated in SFD RPE, suggesting increased ECM turnover. Finally, SFD RPE cells have decreased intracellular reduced glutathione and were found to be more vulnerable to oxidative stress. Our findings suggest that elements of SFD pathology can be demonstrated in culture which may lead to insights into disease mechanisms.

CRISPR Knockouts of pmela and pmelb Engineered a Golden Tilapia by Regulating Relative Pigment Cell Abundance.

Premelanosome protein (pmel) is a key gene for melanogenesis. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study, we found that most fishes have 2 pmel genes arising from the teleost-specific whole-genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypopigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a golden body color. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, although both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.

Systemic biodistribution and hepatocyte-specific gene editing with CRISPR/Cas9 using hyaluronic acid-based nanoparticles.

The goal of this study was to evaluate hepatocyte-specific gene editing, via systemic administration of hyaluronic acid (HA)-based nanoparticles in naïve CD-1 mice. Using HA-poly(ethylene imine) (HA-PEI) and HA-PEI-mannose nanoparticles with differential mannose density (1X and 2X), we have evaluated systemic biodistribution and hepatocyte-specific delivery using IVIS imaging and flow cytometry. Additionally, we have investigated hepatocyte-specific delivery and transfection of CRISPR/Cas9 gene editing plasmid and eGFP gene payload to integrate at the Rosa26 locus. IVIS imaging showed uptake of HA-PEI nanoparticles primarily by the liver, and with addition of mannose at different concentrations, the nanoparticles showed increased uptake in both the liver and spleen. HA-PEI-mannose nanoparticles showed 55-65% uptake by hepatocytes, along with uptake by resident macrophage regardless of the mannose concentration. One of two gRNA targets showed 15% genome editing and obtained similar results for all three nanoparticle formulations. Cells positive for our gene payload were greatest with HA-PEI-mannose-1X nanoparticles where 16.2% of cells were GFP positive. The results were encouraging as proof of concept for the development of a non-viral biodegradable and biocompatible polymeric delivery system for gene editing specifically targeting hepatocytes upon systemic administration.

CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient's quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

Role of Adenylyl Cyclase Type 7 in Functions of BV-2 Microglia.

To assess the role of adenylyl cyclase type 7 (AC7) in microglia's immune function, we generated AC7 gene knockout (AC7 KO) clones from a mouse microglial cell line, BV-2, using the CRISPR-Cas9 gene editing system. The ability of BV-2 cells to generate cAMP and their innate immune functions were examined in the presence or absence of ethanol. The parental BV-2 cells showed robust cAMP production when stimulated with prostaglandin-E1 (PGE1) and ethanol increased cAMP production in a dose-dependent manner. AC7 KO clones of BV-2 cells showed diminished and ethanol-insensitive cAMP production. The phagocytic activity of the parental BV-2 cells was inhibited in the presence of PGE1; AC7 KO BV-2 cells showed lower and PGE1-insensitive phagocytic activity. Innate immune activities of the parental BV-2 cells, including bacterial killing, nitric oxide synthesis, and expression of arginase 1 and interleukin 10 were activated as expected with small effects of ethanol. However, the innate immune activities of AC7 KO cells were either drastically diminished or not detected. The data presented suggest that AC7 has an important role in the innate immune functions of microglial cells. AC7's involvement in ethanol's effects on immune functions remains unclear. Further studies are needed.