Preclinical Evaluation of Foamy Virus Vector-Mediated Gene Addition in Human Hematopoietic Stem/Progenitor Cells for Correction of Leukocyte Adhesion Deficiency Type 1.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the ß2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

N-Acetylcysteine prevents amyloid-ß secretion in neurons derived from human pluripotent stem cells with trisomy 21.

Down syndrome (DS) is caused by the trisomy of chromosome 21. Among the many disabilities found in individuals with DS is an increased risk of early-onset Alzheimer's disease (AD). Although higher oxidative stress and an upregulation of amyloid ß (Aß) peptides from an extra copy of the APP gene are attributed to the AD susceptibility, the relationship between the two factors is unclear. To address this issue, we established an in vitro cellular model using neurons differentiated from DS patient-derived induced pluripotent stem cells (iPSCs) and isogenic euploid iPSCs. Neurons differentiated from DS patient-derived iPSCs secreted more Aß compared to those differentiated from the euploid iPSCs. Treatment of the neurons with an antioxidant, N-acetylcysteine, significantly suppressed the Aß secretion. These findings suggest that oxidative stress has an important role in controlling the Aß level in neurons differentiated from DS patient-derived iPSCs and that N-acetylcysteine can be a potential therapeutic option to ameliorate the Aß secretion.

Liver Injury Increases the Incidence of HCC following AAV Gene Therapy in Mice.

Adeno-associated virus (AAV) integrates into host genomes at low frequency, but when integration occurs in oncogenic hotspots it can cause hepatocellular carcinoma (HCC). Given the possibility of recombinant AAV (rAAV) integration leading to HCC, common causes of liver inflammation like non-alcoholic fatty liver disease (NAFLD) may increase the risk of rAAV-induced HCC. A rAAV targeting the oncogenic mouse Rian locus was used, and as expected led to HCC in all mice infected as neonates, likely due to growth-related hepatocyte proliferation in young mice. Mice infected with rAAV as adults did not develop HCC unless they were fed a diet leading to NAFLD, with increased inflammation and hepatocyte proliferation. Female mice were less susceptible to rAAV-induced HCC, and male mice with NAFLD treated with estrogen exhibited less inflammation and immune exhaustion associated with oncogenesis compared to those without estrogen. Adult NAFLD mice infected with a non-targeted control rAAV also developed HCC, though only half as frequently as those exposed to the Rian targeted rAAV. This study shows that adult mice exposed to rAAV gene therapy in the context of chronic liver disease developed HCC at high frequency, and thus warrants further study in humans given the high prevalence of NAFLD in the population.

Engineering universal cells that evade immune detection.

The prospect of transplanting cells and tissues without the risk of immune rejection or the need for powerful immunosuppressive drugs is the 'holy grail' of transplantation medicine. Now, with the advent of pluripotent stem cells, CRISPR-Cas9 and other gene-editing technologies, the race to create 'off-the-shelf' donor cells that are invisible to the immune system ('universal cells') has started. One important approach for creating such cells involves the manipulation of genes required for immune recognition, in particular HLA class I and II proteins. Other approaches leverage knowledge of immune-cloaking strategies used by certain bacteria, viruses, parasites, the fetus and cancer cells to induce tolerance to allogeneic cell-based therapies by modifying cells to express immune-suppressive molecules such as PD-L1 and CTLA4-Ig. Various academic groups as well as biotechnology and pharmaceutical companies are on the verge of bringing these therapies into the clinic.

Therapeutic discovery for marrow failure with MDS predisposition using pluripotent stem cells.

Monosomy 7 or deletion of 7q (del(7q)) are common clonal cytogenetic abnormalities associated with high grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current non-transplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman Diamond Syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGFß pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild type cells consistent with hyperactivation of the TGFbeta pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGFbeta pathway rescued hematopoiesis in SDS-iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS-predisposition syndrome, and highlight the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.

Lucky, times ten: A career in Texas science.

On January 21, 2017, I received an E-mail from Herb Tabor that I had been simultaneously hoping for and dreading for several years: an invitation to write a "Reflections" article for the Journal of Biological Chemistry On the one hand, I was honored to receive an invitation from Herb, a man I have admired for over 40 years, known for 24 years, and worked with as a member of the Editorial Board and Associate Editor of the Journal of Biological Chemistry for 17 years. On the other hand, the invitation marked the waning of my career as an academic scientist. With these conflicting emotions, I wrote this article with the goals of recording my career history and recognizing the many mentors, trainees, and colleagues who have contributed to it and, perhaps with pretension, with the desire that students who are beginning a career in research will find inspiration in the path I have taken and appreciate the importance of luck.

Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4+ and CD8+ T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers.

Oxysterol Restraint of Cholesterol Synthesis Prevents AIM2 Inflammasome Activation.

Type I interferon restrains interleukin-1ß (IL-1ß)-driven inflammation in macrophages by upregulating cholesterol-25-hydroxylase (Ch25h) and repressing SREBP transcription factors. However, the molecular links between lipid metabolism and IL-1ß production remain obscure. Here, we demonstrate that production of 25-hydroxycholesterol (25-HC) by macrophages is required to prevent inflammasome activation by the DNA sensor protein absent in melanoma 2 (AIM2). We find that in response to bacterial infection or lipopolysaccharide (LPS) stimulation, macrophages upregulate Ch25h to maintain repression of SREBP2 activation and cholesterol synthesis. Increasing macrophage cholesterol content is sufficient to trigger IL-1ß release in a crystal-independent but AIM2-dependent manner. Ch25h deficiency results in cholesterol-dependent reduced mitochondrial respiratory capacity and release of mitochondrial DNA into the cytosol. AIM2 deficiency rescues the increased inflammasome activity observed in Ch25h-/-. Therefore, activated macrophages utilize 25-HC in an anti-inflammatory circuit that maintains mitochondrial integrity and prevents spurious AIM2 inflammasome activation.

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells.

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

Reprint of "Steroid 5α-reductase 2 deficiency".

Dihydrotestosterone is a potent androgen metabolite formed from testosterone by action of 5α-reductase isoenzymes. Mutations in the type 2 isoenzyme cause a disorder of 46,XY sex development, termed 5α-reductase type 2 deficiency and that was described forty years ago. Many mutations in the encoding gene have been reported in different ethnic groups. In affected 46,XY individuals, female external genitalia are common, but Mullerian ducts regress, and the internal urogenital tract is male. Most affected males are raised as females, but virilization occurs at puberty, and male social sex develops thereafter with high frequency. Fertility can be achieved in some affected males with assisted reproduction techniques, and adults with male social sex report a more satisfactory sex life and quality of life as compared to affected individuals with female social sex.

Production and purification of high-titer foamy virus vector for the treatment of leukocyte adhesion deficiency.

Compared to other integrating viral vectors, foamy virus (FV) vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34+ cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(h)CD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ~1.7 × 104 to 1.0 × 106 infectious units (IU)/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized gag, heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1-2 × 109 IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO) concentration, thereby avoiding DMSO-induced toxicity to CD34+ cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.

Steroid 5α-reductase 2 deficiency.

Dihydrotestosterone is a potent androgen metabolite formed from testosterone by action of 5α-reductase isoenzymes. Mutations in the type 2 isoenzyme cause a disorder of 46,XY sex development, termed 5α-reductase type 2 deficiency and that was described forty years ago. Many mutations in the encoding gene have been reported in different ethnic groups. In affected 46,XY individuals, female external genitalia are common, but Mullerian ducts regress, and the internal urogenital tract is male. Most affected males are raised as females, but virilization occurs at puberty, and male social sex develops thereafter with high frequency. Fertility can be achieved in some affected males with assisted reproduction techniques, and adults with male social sex report a more satisfactory sex life and quality of life as compared to affected individuals with female social sex.

Silent IL2RG Gene Editing in Human Pluripotent Stem Cells.

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.

Interdependence between Interleukin-1 and Tumor Necrosis Factor Regulates TNF-Dependent Control of Mycobacterium tuberculosis Infection.

The interleukin-1 receptor I (IL-1RI) is critical for host resistance to Mycobacterium tuberculosis (Mtb), yet the mechanisms of IL-1RI-mediated pathogen control remain unclear. Here, we show that without IL-1RI, Mtb-infected newly recruited Ly6G(hi) myeloid cells failed to upregulate tumor necrosis factor receptor I (TNF-RI) and to produce reactive oxygen species, resulting in compromised pathogen control. Furthermore, simultaneous ablation of IL-1RI and TNF-RI signaling on either stroma or hematopoietic cells led to early lethality, indicating non-redundant and synergistic roles of IL-1 and TNF in mediating macrophage-stroma cross-talk that was critical for optimal control of Mtb infection. Finally, we show that even in the presence of functional Mtb-specific adaptive immunity, the lack of IL-1α and not IL-1ß led to an exuberant intracellular pathogen replication and progressive non-resolving inflammation. Our study reveals functional interdependence between IL-1 and TNF in enabling Mtb control mechanisms that are critical for host survival.

Adeno-associated virus finds its disease.

Adeno-associated virus (AAV) vectors have been widely adopted for use in gene therapy. A new study raises concerns regarding this approach, reporting that chromosomal insertions of AAV serotype 2 seem to activate proto-oncogenes in human hepatocellular carcinoma.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic.

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

A genome-wide map of adeno-associated virus-mediated human gene targeting.

To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. We used an adeno-associated virus vector to target identical loci introduced as transcriptionally active retroviral vectors. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats or DNase I-hypersensitive sites. Targeted sites were preferentially located within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. We determined the impact of DNA replication by mapping replication forks, which revealed a preference for recombination at target loci transcribed toward an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells and demonstrate a strong recombinogenic effect of colliding polymerases.

Genetic, anatomic, and clinical determinants of human serum sterol and vitamin D levels.

An unknown fraction of the genome participates in the metabolism of sterols and vitamin D, two classes of lipids with diverse physiological and pathophysiological roles. Here, we used mass spectrometry to measure the abundance of >60 sterol and vitamin D derivatives in 3,230 serum samples from a well-phenotyped patient population. Twenty-nine of these lipids were detected in a majority of samples at levels that varied over thousands of fold in different individuals. Pairwise correlations between sterol and vitamin D levels revealed evidence for shared metabolic pathways, additional substrates for known enzymes, and transcriptional regulatory networks. Serum levels of multiple sterols and vitamin D metabolites varied significantly by sex, ethnicity, and age. A genome-wide association study identified 16 loci that were associated with levels of 19 sterols and 25-hydroxylated derivatives of vitamin D (P < 10(-7)). Resequencing, expression analysis, and biochemical experiments focused on one such locus (CYP39A1), revealed multiple loss-of-function alleles with additive effects on serum levels of the oxysterol, 24S-hydroxycholesterol, a substrate of the encoded enzyme. Body mass index, serum lipid levels, and hematocrit were strong phenotypic correlates of interindividual variation in multiple sterols and vitamin D metabolites. We conclude that correlating population-based analytical measurements with genotype and phenotype provides productive insight into human intermediary metabolism.

Inflammation. 25-Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I interferon.

Type I interferon (IFN) protects against viruses, yet it also has a poorly understood suppressive influence on inflammation. Here, we report that activated mouse macrophages lacking the IFN-stimulated gene cholesterol 25-hydroxylase (Ch25h) and that are unable to produce the oxysterol 25-hydroxycholesterol (25-HC) overproduce inflammatory interleukin-1 (IL-1) family cytokines. 25-HC acts by antagonizing sterol response element-binding protein (SREBP) processing to reduce Il1b transcription and to broadly repress IL-1-activating inflammasomes. In accord with these dual actions of 25-HC, Ch25h-deficient mice exhibit increased sensitivity to septic shock, exacerbated experimental autoimmune encephalomyelitis, and a stronger ability to repress bacterial growth. These findings identify an oxysterol, 25-HC, as a critical mediator in the negative-feedback pathway of IFN signaling on IL-1 family cytokine production and inflammasome activity.