Liver-directed SERPINA1 gene therapy attenuates progression of spontaneous and tobacco smoke-induced emphysema in α1-antitrypsin null mice.

α1-antitrypsin deficiency is a rare genetic condition that can cause liver and/or lung disease. There is currently no cure for this disorder, although repeated infusions of plasma-purified protein may slow down emphysema progression. Gene therapy in which a single recombinant adeno-associated viral vector (rAAV) administration would lead to sustained protein expression could therefore similarly affect disease progression, and provide the added benefits of reducing treatment burden and thereby improving the patient's quality of life. The study presented here tests whether treating the Serpina1a-e knockout mouse model of α1-antitrypsin-deficiency lung disease with gene therapy would have an impact on the disease course, either on spontaneous disease caused by aging or on accelerated disease caused by exposure to cigarette smoke. Liver-directed gene therapy led to dose-dependent levels of biologically active human α1-antitrypsin protein. Furthermore, decreased lung compliance and increased elastic recoil indicate that treated mice had largely preserved lung tissue elasticity and alveolar wall integrity compared with untreated mice. rAAV-mediated gene augmentation is therefore able to compensate for the loss of function and restore a beneficial lung protease-antiprotease balance. This work constitutes a preclinical study report of a disease-modifying treatment in the Serpina1a-e knockout mouse model using a liver-specific rAAV serotype 8 capsid.

Evaluation of cytosine base editing and adenine base editing as a potential treatment for alpha-1 antitrypsin deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a rare autosomal codominant disease caused by mutations within the SERPINA1 gene. The most prevalent variant in patients is PiZ SERPINA1, containing a single G > A transition mutation. PiZ alpha-1 antitrypsin (AAT) is prone to misfolding, leading to the accumulation of toxic aggregates within hepatocytes. In addition, the abnormally low level of AAT secreted into circulation provides insufficient inhibition of neutrophil elastase within the lungs, eventually causing emphysema. Cytosine and adenine base editors enable the programmable conversion of C⋅G to T⋅A and A⋅T to G⋅C base pairs, respectively. In this study, two different base editing approaches were developed: use of a cytosine base editor to install a compensatory mutation (p.Met374Ile) and use of an adenine base editor to mediate the correction of the pathogenic PiZ mutation. After treatment with lipid nanoparticles formulated with base editing reagents, PiZ-transgenic mice exhibited durable editing of SERPINA1 in the liver, increased serum AAT, and improved liver histology. These results indicate that base editing has the potential to address both lung and liver disease in AATD.

Muscle-Directed Delivery of an AAV1 Vector Leads to Capsid-Specific T Cell Exhaustion in Nonhuman Primates and Humans.

With the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals for Zolgensma, Luxturna, and Glybera, recombinant adeno-associated viruses (rAAVs) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsids and/or transgenes. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM delivery, venous limb perfusion, and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both day 21 and day 60 post-injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post-vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells.

Intralingual and Intrapleural AAV Gene Therapy Prolongs Survival in a SOD1 ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results in death from respiratory failure. No cure exists for this devastating disease, but therapy that directly targets the respiratory system has the potential to prolong survival and improve quality of life in some cases of ALS. The objective of this study was to enhance breathing and prolong survival by suppressing superoxide dismutase 1 (SOD1) expression in respiratory motor neurons using adeno-associated virus (AAV) expressing an artificial microRNA targeting the SOD1 gene. AAV-miRSOD1 was injected in the tongue and intrapleural space of SOD1G93A mice, and repetitive respiratory and behavioral measurements were performed until the end stage. Robust silencing of SOD1 was observed in the diaphragm and tongue as well as systemically. Silencing of SOD1 prolonged survival by approximately 50 days, and it delayed weight loss and limb weakness in treated animals compared to untreated controls. Histologically, there was preservation of the neuromuscular junctions in the diaphragm as well as the number of axons in the phrenic and hypoglossal nerves. Although SOD1 suppression improved breathing and prolonged survival, it did not ameliorate the restrictive lung phenotype. Suppression of SOD1 expression in motor neurons that underlie respiratory function prolongs survival and enhances breathing until the end stage in SOD1G93A ALS mice.

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.

Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)1 and Hermansky-Pudlak syndrome type 1 (HPS1)2. Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.

AAV9 gene replacement therapy for respiratory insufficiency in very-long chain acyl-CoA dehydrogenase deficiency.

Very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an autosomal recessive disorder of fatty acid oxidation. Fatty acids are a major source of energy during catabolic stress, so the absence of VLCAD can result in a metabolic crises and respiratory insufficiency. The etiology of this respiratory insufficiency is unclear. Thus, our aims were: (1) to characterize respiratory pathophysiology in VLCADD mice (VLCAD-/- ), and (2) to determine if AAV9-mediated gene therapy improves respiratory function. For the first aim, VLCAD-/- and wild-type (WT) mice underwent an exercise/fast "stress protocol" and awake spontaneous breathing was evaluated using whole-body plethysmography (WBP) both at baseline and during a hypercapnic respiratory challenge (FiO2 : 0.21; FiCO2 : 0.07; nitrogen balance). During hypercapnia, VLCAD -/- mice had a significantly lower frequency, tidal volume, minute ventilation, and peak inspiratory and expiratory flow, all of which indicate respiratory insufficiency. Histologically, the cardiac and respiratory muscles of stressed VLCAD -/- animals had an accumulation of intramyocellular lipids. For the second aim, a single systemic injection of AAV9-VLCAD gene therapy improved this respiratory pathology by normalizing breathing frequency and enhancing peak inspiratory flow. In addition, following gene therapy, there was a moderate reduction of lipid accumulation in the respiratory muscles. Furthermore, VLCAD protein expression was robust in cardiac and respiratory muscle. This was confirmed by immuno-staining with anti-human VLCAD antibody. In summary, stress with exercise and fasting induces respiratory insufficiency in VLCAD-/- mice and a single injection with AAV9-VLCAD gene therapy ameliorates breathing.

Systemic Delivery of AAVB1-GAA Clears Glycogen and Prolongs Survival in a Mouse Model of Pompe Disease.

Pompe disease is an autosomal recessive glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption in muscle and the central nervous system (CNS). Adeno-associated virus (AAV) gene therapy is ideal for Pompe disease, since a single systemic injection may correct both muscle and CNS pathologies. Using the Pompe mouse (B6;129-GaaTm1Rabn/J), this study sought to explore if AAVB1, a newly engineered vector with a high affinity for muscle and CNS, reduces systemic weakness and improves survival in adult mice. Three-month-old Gaa-/- animals were injected with either AAVB1 or AAV9 vectors expressing GAA and tissues were harvested 6 months later. Both AAV vectors prolonged survival. AAVB1-treated animals had a robust weight gain compared to the AAV9-treated group. Vector genome levels, GAA enzyme activity, and histological analysis indicated that both vectors transduced the heart efficiently, leading to glycogen clearance, and transduced the diaphragm and CNS at comparable levels. AAVB1-treated mice had higher GAA activity and greater glycogen clearance in the tongue. Finally, AAVB1-treated animals showed improved respiratory function comparable to wild-type animals. In conclusion, AAVB1-GAA offers a promising therapeutic option for the treatment of muscle and CNS in Pompe disease.

Aerobic Glycolysis Is Essential for Normal Rod Function and Controls Secondary Cone Death in Retinitis Pigmentosa.

Aerobic glycolysis accounts for ∼80%-90% of glucose used by adult photoreceptors (PRs); yet, the importance of aerobic glycolysis for PR function or survival remains unclear. Here, we further established the role of aerobic glycolysis in murine rod and cone PRs. We show that loss of hexokinase-2 (HK2), a key aerobic glycolysis enzyme, does not affect PR survival or structure but is required for normal rod function. Rods with HK2 loss increase their mitochondrial number, suggesting an adaptation to the inhibition of aerobic glycolysis. In contrast, cones adapt without increased mitochondrial number but require HK2 to adapt to metabolic stress conditions such as those encountered in retinitis pigmentosa, where the loss of rods causes a nutrient shortage in cones. The data support a model where aerobic glycolysis in PRs is not a necessity but rather a metabolic choice that maximizes PR function and adaptability to nutrient stress conditions.

Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema.

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is α-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a-e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research.

Airway smooth muscle dysfunction in Pompe (Gaa-/- ) mice.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons, and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease, the Gaa-/- mouse, we compared histology, pulmonary mechanics, airway smooth muscle (ASM) function, and calcium signaling between Gaa-/- and age-matched wild-type (WT) mice. Lysosomal glycogen accumulation was observed in the smooth muscle of both the bronchi and the trachea in Gaa-/- but not WT mice. Furthermore, Gaa-/- mice had hyporesponsive airway resistance and bronchial ring contraction to the bronchoconstrictive agents methacholine (MCh) and potassium chloride (KCl) and to a bronchodilator (albuterol). Finally, calcium signaling during bronchiolar smooth muscle contraction was impaired in Gaa-/- mice indicating impaired extracellular calcium influx. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the trachea and bronchi and impairs the ability of lower ASM to regulate calcium and respond appropriately to bronchodilator or constrictors. Accordingly, ASM dysfunction may contribute to respiratory impairments in Pompe disease.

Improved cell metabolism prolongs photoreceptor survival upon retinal-pigmented epithelium loss in the sodium iodate induced model of geographic atrophy.

Age-related macular degeneration (AMD) is characterized by malfunction and loss of retinal-pigmented epithelium (RPE) cells. Because the RPE transfers nutrients from the choriocapillaris to photoreceptor (PR), PRs are affected as well. Geographic atrophy (GA) is an advanced form of AMD characterized by severe vision impairment due to RPE loss over large areas. Currently there is no treatment to delay the degeneration of nutrient deprived PRs once RPE cells die. Here we show that cell-autonomous activation of the key regulator of cell metabolism, the kinase mammalian target of rapamycin complex 1 (mTORC1), delays PR death in the sodium iodate induced model of RPE atrophy. Consistent with this finding loss of mTORC1 in cones accelerates cone death as cones fail to balance demand with supply. Interestingly, promoting rod survival does not promote cone survival in this model of RPE atrophy as both, rods and cones suffer from a sick and dying RPE. The findings suggest that activation of metabolic genes downstream of mTORC1 can serve as a strategy to prolong PR survival when RPE cells malfunction or die.

CX3CL1 (fractalkine) protein expression in normal and degenerating mouse retina: in vivo studies.

We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated ∼100 kDa CX3CL1 form and a cleaved ∼85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal ∼95 kDa form and a decrease in the ∼85-kDa form were observed. From P14, a ∼95 kDa form became principal in wt retina, while in rd10 retinas a soluble ∼85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1.