CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.

BACKGROUND: Sickle cell disease is caused by a defect in the ß-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny. METHODS: We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923. RESULTS: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period. CONCLUSIONS: CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).

A molecular glue degrader of the WIZ transcription factor for fetal hemoglobin induction.

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.

Muscle-specific expression of PPARgamma coactivator-1alpha improves exercise performance and increases peak oxygen uptake.

The induction of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a key regulator of mitochondriogenesis, is well-established under multiple physical exercise regimens, including, endurance, resistance, and sprint training. We wanted to determine if increased expression of PGC-1alpha in muscle is sufficient to improve performance during exercise in vivo. We demonstrate that muscle-specific expression of PGC-1alpha improves the performance during voluntary as well as forced exercise challenges. Additionally, PGC-1alpha transgenic mice exhibit an enhanced performance during a peak oxygen uptake exercise test, demonstrating an increased peak oxidative capacity, or whole body oxygen uptake. This increased ability to perform in multiple exercise paradigms is supported by enhanced mitochondrial function as suggested by increased mitochondrial gene expression, mitochondrial DNA, and mitochondrial enzyme activity. Thus this study demonstrates that upregulation of PGC-1alpha in muscle in vivo is sufficient to greatly improve exercise performance under various exercise paradigms as well as increase peak oxygen uptake.

TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.

Gene therapy with inducible nitric oxide synthase protects against myocardial infarction via a cyclooxygenase-2-dependent mechanism.

Although the inducible isoform of NO synthase (iNOS) mediates late preconditioning (PC), it is unknown whether iNOS gene transfer can replicate the cardioprotective effects of late PC, and the role of this protein in myocardial ischemia is controversial. Thus, the cDNA for human iNOS was cloned behind the Rous sarcoma virus (RSV) promoter to create adenovirus (Ad) 5/iNOS lacking E1, E2a, and E3 regions. Intramyocardial injection of Ad5/iNOS in mice increased local iNOS protein expression and activity and markedly reduced infarct size. The infarct-sparing effects of Ad5/iNOS were at least as powerful as those of ischemic PC. The increased iNOS expression was associated with increased cyclooxygenase-2 (COX-2) protein expression and prostanoid levels. Pretreatment with the COX-2-selective inhibitor NS-398 completely abrogated the infarct-sparing actions of Ad5/iNOS, demonstrating that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection. We conclude that gene transfer of iNOS (an enzyme commonly thought to be detrimental) affords powerful cardioprotection the magnitude of which is equivalent to that of late PC. This is the first report that upregulation of iNOS, in itself, is sufficient to reduce infarct size. The results provide proof-of-principle for gene therapy against ischemia/reperfusion injury, which increases local myocardial NO synthase levels without the need for continuous intravenous infusion of NO donors and without altering systemic hemodynamics. The data also reveal the existence of a close coupling between iNOS and COX-2, whereby induction of the former enzyme leads to secondary induction of the latter, which in turn mediates the cytoprotective effects of iNOS. We propose that iNOS and COX-2 form a stress-responsive functional module that mitigates ischemia/reperfusion injury.

Overexpression of soluble fas attenuates transplant arteriosclerosis in rat aortic allografts.

BACKGROUND: The killing of vascular cells by activated macrophages is an important step in the process of destabilization of the arterial wall. The death receptor Fas is implicated in vascular cell death. Hence, we extended our studies in a rat aortic allograft model, using adenovirus-mediated overexpression of soluble Fas (sFas) to block Fas binding to Fas ligand (Fas-L). The contribution of Fas to vascular cell injury and consequent transplant arteriosclerosis was investigated. METHODS AND RESULTS: Activated monocytes in the presence of macrophage colony-stimulating factor induce endothelial cell apoptosis in vitro, which was significantly inhibited by adenovirus-mediated sFas overexpression. Next, donor rat abdominal aortas were either untreated or transduced with adenoviruses encoding (1) rat soluble Fas (Ad3rsFas), (2) no insert (Ad3Null), and (3) beta-galactosidase (Ad3nBg). A total of 175 aortic grafts were harvested 2 to 90 days after transplantation. Vascular cell apoptosis and CD45+ cell infiltration were significantly reduced in Ad3rsFas-transduced aortas, as compared with control allografts. Moreover, the control allografts developed marked intimal thickening, whereas Ad3rsFas-transduced allografts had significantly less neointima until the 90-day time point. CONCLUSIONS: sFas overexpression protects the integrity of the vessel wall from immune injury and attenuates transplant arteriosclerosis.

Inhibition of accelerated graft arteriosclerosis by gene transfer of soluble fibroblast growth factor receptor-1 in rat aortic transplants.

OBJECTIVE: Because increased fibroblast growth factor-1 (FGF-1) and FGF receptor (FGFR) expression correlate with the development of accelerated graft arteriosclerosis in transplanted human hearts, this study sought to determine whether local gene transfer of soluble FGFR-1, capable of binding both FGF-1 and FGF-2, could blunt the development of accelerated graft arteriosclerosis in the rat aortic transplant model. METHODS AND RESULTS: A construct encoding the FGFR-1 ectodomain, capable of neutralizing FGF-2 action, was expressed in rat aortic allografts, using adenoviral gene transfer at the time of transplantation. Neointima formation was inhibited in aortic allografts transduced with soluble FGFR-1, compared with allografts transduced with Null virus. CONCLUSIONS: FGFs play a causal role in the development of accelerated graft arteriosclerosis in the rat aortic transplant model. Targeted interruption of FGF function could potentially reduce neointima formation in patients with heart and kidney transplants.

Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice.

Adenoviral vectors used in gene therapy are predominantly derived from adenovirus serotype 5 (Ad5), which infects a broad range of cells. Ad5 cell entry involves interactions with the coxsackie-adenovirus receptor (CAR) and integrins. To assess these receptors in vivo, we mutated amino acid residues in fiber and penton that are involved in receptor interaction and showed that CAR and integrins play a minor role in hepatic transduction but that integrins can influence gene delivery to other tissues. These data suggest that an alternative entry pathway exists for hepatocyte transduction in vivo that is more important than CAR or integrins. In vitro data suggest a role for heparan sulfate glycosaminoglycans (HSG) in adenovirus transduction. The role of the fiber shaft in liver uptake was examined by introducing specific amino acid changes into a putative HSG-binding motif contained within the shaft or by preparing fiber shaft chimeras between Ad5 and Ad35 fibers. Results were obtained that demonstrate that the Ad5 fiber shaft can influence gene transfer both in vitro and to the liver in vivo. These observations indicate that the currently accepted two-step entry pathway, which involves CAR and integrins, described for adenoviral infection in vitro, is not used for hepatic gene transfer in vivo. In contrast, alpha(v) integrins influence gene delivery to the lung, spleen, heart, and kidney. The detargeted vector constructs described here may provide a foundation for the development of targeted adenoviral vectors.

Receptor interactions involved in adenoviral-mediated gene delivery after systemic administration in non-human primates.

Adenovirus serotype 5 (Ad5)-based vectors can bind at least three separate cell surface receptors for efficient cell entry: the coxsackie-adenovirus receptor (CAR), alpha nu integrins, and heparan sulfate glycosaminoglycans (HSG). To address the role of each receptor involved in adenoviral cell entry, we mutated critical amino acids in fiber or penton to inhibit receptor interaction. A series of five adenoviral vectors was prepared and the biodistribution of each was previously characterized in mice. To evaluate possible species differences in Ad vector tropism, we characterized the effects of each detargeting mutation in non-human primates after systemic delivery to confirm our conclusions made in mice. In non-human primates, CAR was found to have minimal effects on vector delivery to all organs examined including liver and spleen. Cell-surface alpha nu integrins played a significant role in delivery of vector to the spleen, lung and kidney. The fiber shaft mutation S*, which presumably inhibits HSG binding, was found to significantly decrease delivery to all organs examined. The ability to detarget the liver corresponded with decreased elevations in liver serum enzymes (aspartate transferase [AST] and alanine transferase [ALT]) 24 hr after vector administration and also in serum interleukin (IL)-6 levels 6 hr after vector administration. The biodistribution data generated in cynomolgus monkeys correspond with those data derived from mice, demonstrating that CAR binding is not the major determinant of viral tropism in vivo. Vectors containing the fiber shaft modification may provide for a detargeted adenoviral vector on which to introduce new tropisms for the development of targeted, systemically deliverable adenoviral vectors for human clinical application.

Interaction of systemically delivered adenovirus vectors with Kupffer cells in mouse liver.

When adenovirus (Ad) vectors are injected intravenously they are rapidly taken up by Kupffer cells (KCs) in the liver. This results in massive KC necrosis within minutes, followed by a more gradual disappearance of KCs from the liver. It is not known how KCs recognize Ad, or why Ad kills KCs. We used a variety of mutated and fiber-pseudotyped Ad vectors to evaluate how capsid proteins influence Ad uptake by KCs and to define the viral proteins that are involved in the destruction of KCs. We found that depletion of KCs from the liver was partially dependent on interactions between Ad and integrins, but was independent of the coxsackievirus and Ad receptor. The Ad5 fiber shaft was proven to be a particularly important contributory factor, because vectors with the shorter Ad35 shaft were not as effective at depleting KCs. In contrast, the fiber head played no discernible role. Variations in the ability of Ad vectors to deplete KCs could not be explained by differences in the amount of Ad that reached KCs, because all mutant Ads were accumulated by KCs at similar levels. Interestingly, we found that the Ad mutant ts1 did not cause KC death; this virus is known to bind and enter cells normally, but the capsid is unable to disassemble or lyse membranes. We conclude that Ad vectors kill KCs at a postbinding step and that this cell death can be mitigated if downstream events in viral entry are blocked.

Identification of novel modulators of mitochondrial function by a genome-wide RNAi screen in Drosophila melanogaster.

Mitochondrial dysfunction is associated with many human diseases. There has not been a systematic genetic approach for identifying regulators of basal mitochondrial biogenesis and function in higher eukaryotes. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells using mitochondrial Citrate synthase (CS) activity as the primary readout. We screened 13,071 dsRNAs and identified 152 genes that modulate CS activity. These modulators are involved in a wide range of biological processes and pathways including mitochondrial-related functions, transcriptional and translational regulation, and signaling pathways. Selected hits among the 152 genes were further analyzed for their effect on mitochondrial CS activity in transgenic flies or fly mutants. We confirmed a number of gene hits including HDAC6, Rpd3(HDAC1), CG3249, vimar, Src42A, klumpfuss, barren, and smt3 which exert effects on mitochondrial CS activities in vivo, demonstrating the value of Drosophila genome-wide RNAi screens for identifying genes and pathways that modulate mitochondrial function.

Estrogen-related receptor alpha is essential for the expression of antioxidant protection genes and mitochondrial function.

Estrogen-related receptor alpha (ERRalpha) is an important mediator of mitochondrial biogenesis and function. To investigate the transcriptional network controlling these phenomena, we investigated mitochondrial gene expression in embryonic fibroblasts isolated from ERRalpha null mice. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) stimulated mitochondrial gene expression program in control cells, but not in the ERRalpha null cells. Interestingly, the induction of levels of mitochondrial oxidative stress protection genes in response to increased PGC-1alpha levels was dependent on ERRalpha. Furthermore, we found that the PGC-1alpha-mediated induction of estrogen-related receptor gamma and nuclear respiratory factor 2 (NRF-2), was dependent on the presence of ERRalpha. Basal levels of NRF-2 were decreased in the absence of ERRalpha. The absence of ERRalpha resulted in a decrease in citrate synthase enzyme activity in response to PGC-1alpha overexpression. Our results indicate an essential role for ERRalpha as a key regulator of oxidative metabolism.

Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1alpha transcription and mitochondrial biogenesis in muscle cells.

PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha) is a master regulator of mitochondrial biogenesis and plays an important role in several other aspects of energy metabolism. To identify upstream regulators of PGC-1alpha gene transcription, 10,000 human full-length cDNAs were screened for induction of the PGC-1alpha promoter. A number of activators of PGC-1alpha transcription were found; the most potent activator was the transducer of regulated CREB (cAMP response element-binding protein) binding protein (TORC) 1, a coactivator of CREB. The other two members of the TORC family, TORC2 and TORC3, also strongly activated PGC-1alpha transcription. TORCs dramatically induced PGC-1alpha gene transcription through CREB. Forced expression of TORCs in primary muscle cells induced the endogenous mRNA of PGC-1alpha and its downstream target genes in the mitochondrial respiratory chain and TCA cycle. Importantly, these changes in gene expression resulted in increased mitochondrial oxidative capacity measured by cellular respiration and fatty acid oxidation. Finally, we demonstrated that the action of TORCs in promoting mitochondrial gene expression and function requires PGC-1alpha. Previous studies had indicated that TORCs function as a calcium- and cAMP-sensitive coincidence detector and mediate individual and synergistic effects of these two pathways. Our results, together with previous findings, strongly suggest that TORCs play a key role in linking these external signals to the transcriptional program of adaptive mitochondrial biogenesis by activating PGC-1alpha gene transcription.

In vivo hepatic adenoviral gene delivery occurs independently of the coxsackievirus-adenovirus receptor.

Systemic administration of adenoviral vectors leads to a widespread distribution of vector. Therefore, targeting of adenoviral vectors to specific tissues or cell types will require methods to ablate the normal tropism of the vector simultaneously with the introduction of new receptor specificities. To inhibit native receptor binding, we mutated residues in the AB loop of the adenovirus type 5 (Ad5) fiber. We genetically incorporated the S408E-P409A mutation, referred to as KO1, into the adenoviral genome alone or in combination with an RGD-targeting ligand in the HI loop of fiber. Transduction experiments confirmed that the KO1 mutation results in a significant reduction in fiber-dependent gene transfer on A549 and primary fibroblast cells that could be restored via the RGD-targeting ligand. Competition transduction experiments verified the receptor-binding properties of each vector on A549 and hepatocytes in vitro. Unexpectedly, in mice systemic delivery of the vector containing the KO1 mutation resulted in efficient liver transduction that was localized specifically to hepatocytes. We confirmed these results in three different mouse strains, indicating that hepatic adenoviral gene transfer may be independent of the coxsackievirus-adenovirus receptor and that in vivo retargeting will require further viral capsid modifications to generate a fully detargeted adenoviral vector upon which to introduce new tropisms.

In vivo transduction of photoreceptors or ciliary body by intravitreal injection of pseudotyped adenoviral vectors.

Strategies for retargeting adenoviral (Ad) vectors have been developed, but their in vivo efficacy remains to be demonstrated. Gene delivery to specific ocular cell types represents an approach to treating many diseases that cause irreversible blindness. One of these cell types, the photoreceptor (PR), is not infected by standard Ad5-based vectors. We evaluated gene delivery after intraocular injection of Ads pseudotyped with three different fiber proteins and found three distinct patterns of infection. An intravitreally injected Ad5 vector readily infected the iris, corneal endothelium, and ciliary body, while few cells in the retina expressed transgene product. In contrast, an Ad3-pseudotyped virus selectively transduced ciliary body, of interest for treating diseases such as glaucoma. A vector pseudotyped with the fiber protein of Ad37 transduced PRs as well as ciliary body. This finding has potential application to the treatment of retinal degenerative or neovascular diseases. These studies demonstrate cell type-selective gene delivery in vivo with retargeted Ads, provide information about the cellular tropisms of several Ad serotypes, and should lead to improved strategies for preserving vision.

Targeting adenoviral vectors by using the extracellular domain of the coxsackie-adenovirus receptor: improved potency via trimerization.

Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo.

Effect of adenovirus serotype 5 fiber and penton modifications on in vivo tropism in rats.

Sequestration of adenovirus serotype 5 (Ad5) in liver restricts its use for gene delivery to other target sites in vivo. To date, no studies have systematically assessed the impact of genetic capsid modifications on in vivo tropism in rats, an important preclinical model for many disease types. We evaluated a panel of Ad5 vectors with capsid mutations or pseudotyped with the short fiber from serotype 41 (Ad41s) for infectivity in Wistar Kyoto rats in vitro and systemically in vivo. In vitro studies demonstrated that both coxsackie and adenovirus receptor (CAR) and heparan sulfate proteoglycan (HSPG) binding were predominant predictors of Ad5 tropism. In vivo, neither CAR nor integrin mutations alone affected liver transduction. The HSPG-binding mutation alone moderately reduced rat liver transgene levels by 2-fold (P < 0.05). This was further substantially decreased by additional mutation of CAR binding (95-fold). Combining CAR and integrin mutations reduced transgene levels by >99% (509-fold, P < 0.01), an effect not observed in parallel experiments in mice and highly variable when studied further in an additional two strains of rat. Ad41s mediated very low liver transduction (58-fold lower than AdCTL). Moreover, CAR-binding mutants (KO1-containing) or pseudotyping 41s eliminated hemagglutination of rat and human red blood cells in vitro. This highlights some important potential species and strain differences dictating Ad5 tropism in vivo and identifies vectors that are substantially detargeted from rat liver in vivo.