Viral Vector-mediated Gene Transfer Research Articles

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa−/−) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa−/− mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa−/− mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa−/− mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa−/− mouse model via liver gene transfer of secGAA.

Viral Vector-Mediated Gene Transfer of Glutamic Acid Decarboxylase for Chronic Pain Treatment: A Literature Review.

Chronic pain is long-lasting nociceptive state, impairing the patient's quality of life. Existing analgesics are generally not effective in the treatment of chronic pain, some of which such as opioids have the risk of tolerance/dependence and overdose death with higher daily opioid doses for increasing analgesic effect. Opioid use disorders have already reached an epidemic level in the United States; therefore, nonopioid analgesic approach and/or use of nonpharmacologic interventions will be employed with increasing frequency. Viral vector-mediated gene therapy is promising in clinical trials in the nervous system diseases. Glutamic acid decarboxylase (GAD) enzyme, a key enzyme in biosynthesis of γ-aminobutyric acid (GABA), plays an important role in analgesic mechanism. In the literature review, we used PubMed and bioRxiv to search the studies, and the eligible criteria include (1) article written in English, (2) use of viral vectors expressing GAD67 or GAD65, and (3) preclinical pain models. We identified 13 eligible original research articles, in which the pain models include nerve injury, HIV-related pain, painful diabetic neuropathy, and formalin test. GAD expressed by the viral vectors from all the reports produced antinociceptive effects. Restoring GABA systems is a promising therapeutic strategy for chronic pain, which provides evidence for the clinical trial of gene therapy for pain in the near future.

Development of a gene therapy for Fabry disease using adeno-associated viral vector mediated gene transfer

Development of a gene therapy for Fabry disease using adeno-associated viral vector mediated gene transfer

Cytokine mRNA Degradation in Cardiomyocytes Restrains Sterile Inflammation in Pressure-Overloaded Hearts.

Proinflammatory cytokines play an important role in the pathogenesis of heart failure. The mechanisms responsible for maintaining sterile inflammation within failing hearts remain poorly defined. Although transcriptional control is important for proinflammatory cytokine gene expression, the stability of mRNA also contributes to the kinetics of immune responses. Regnase-1 is an RNase involved in the degradation of a set of proinflammatory cytokine mRNAs in immune cells. The role of Regnase-1 in nonimmune cells such as cardiomyocytes remains to be elucidated. To examine the role of proinflammatory cytokine degradation by Regnase-1 in cardiomyocytes, cardiomyocyte-specific Regnase-1-deficient mice were generated. The mice were subjected to pressure overload by means of transverse aortic constriction to induce heart failure. Cardiac remodeling was assessed by echocardiography as well as histological and molecular analyses 4 weeks after operation. Inflammatory cell infiltration was examined by immunostaining. Interleukin-6 signaling was inhibited by administration with its receptor antibody. Overexpression of Regnase-1 in the heart was performed by adeno-associated viral vector-mediated gene transfer. Cardiomyocyte-specific Regnase-1-deficient mice showed no cardiac phenotypes under baseline conditions, but exhibited severe inflammation and dilated cardiomyopathy after 4 weeks of pressure overload compared with control littermates. Four weeks after transverse aortic constriction, the Il6 mRNA level was upregulated, but not other cytokine mRNAs, including tumor necrosis factor-α, in Regnase-1-deficient hearts. Although the Il6 mRNA level increased 1 week after operation in both Regnase-1-deficient and control hearts, it showed no increase in control hearts 4 weeks after operation. Administration of anti-interleukin-6 receptor antibody attenuated the development of inflammation and cardiomyopathy in cardiomyocyte-specific Regnase-1-deficient mice. In severe pressure overloaded wild-type mouse hearts, sustained induction of Il6 mRNA was observed, even though the protein level of Regnase-1 increased. Adeno-associated virus 9-mediated cardiomyocyte-targeted gene delivery of Regnase-1 or administration of anti-interleukin-6 receptor antibody attenuated the development of cardiomyopathy induced by severe pressure overload in wild-type mice. The degradation of cytokine mRNA by Regnase-1 in cardiomyocytes plays an important role in restraining sterile inflammation in failing hearts and the Regnase-1-mediated pathway might be a therapeutic target to treat patients with heart failure.

Intracrine Endorphinergic Systems in Modulation of Myocardial Differentiation.

A wide variety of peptides not only interact with the cell surface, but govern complex signaling from inside the cell. This has been referred to as an “intracrine” action, and the orchestrating molecules as “intracrines”. Here, we review the intracrine action of dynorphin B, a bioactive end-product of the prodynorphin gene, on nuclear opioid receptors and nuclear protein kinase C signaling to stimulate the transcription of a gene program of cardiogenesis. The ability of intracrine dynorphin B to prime the transcription of its own coding gene in isolated nuclei is discussed as a feed-forward loop of gene expression amplification and synchronization. We describe the role of hyaluronan mixed esters of butyric and retinoic acids as synthetic intracrines, controlling prodynorphin gene expression, cardiogenesis, and cardiac repair. We also discuss the increase in prodynorphin gene transcription and intracellular dynorphin B afforded by electromagnetic fields in stem cells, as a mechanism of cardiogenic signaling and enhancement in the yield of stem cell-derived cardiomyocytes. We underline the possibility of using the diffusive features of physical energies to modulate intracrinergic systems without the needs of viral vector-mediated gene transfer technologies, and prompt the exploration of this hypothesis in the near future.

In Vivo Gene Therapy for Mucopolysaccharidosis Type III (Sanfilippo Syndrome): A New Treatment Horizon.

For most lysosomal storage diseases (LSDs), there is no cure. Gene therapy is an attractive tool for treatment of LSDs caused by deficiencies in secretable lysosomal enzymes, in which neither full restoration of normal enzymatic activity nor transduction of all cells of the affected organ is necessary. However, some LSDs, such as mucopolysaccharidosis type III (MPSIII) diseases or Sanfilippo syndrome, represent a difficult challenge because patients suffer severe neurodegeneration with mild somatic alterations. The disease's main target is the central nervous system (CNS) and enzymes do not efficiently cross the blood-brain barrier (BBB) even if present at very high concentration in circulation. No specific treatment has been approved for MPSIII. In this study, we discuss the adeno-associated virus (AAV) vector-mediated gene transfer strategies currently being developed for MPSIII disease. These strategies rely on local delivery of AAV vectors to the CNS either through direct intraparenchymal injection at several sites or through delivery to the cerebrospinal fluid (CSF), which bathes the whole CNS, or exploit the properties of certain AAV serotypes capable of crossing the BBB upon systemic administration. Although studies in small and large animal models of MPSIII diseases have provided evidence supporting the efficacy and safety of all these strategies, there are considerable differences between the different routes of administration in terms of procedure-associated risks, vector dose requirements, sensitivity to the effect of circulating neutralizing antibodies that block AAV transduction, and potential toxicity. Ongoing clinical studies should shed light on which gene transfer strategy leads to highest clinical benefits while minimizing risks. The development of all these strategies opens a new horizon for treatment of not only MPSIII and other LSDs but also of a wide range of neurological diseases.

LAV-BPIFB4 associates with reduced frailty in humans and its transfer prevents frailty progression in old mice

Background: There is an increasing concern about age-related frailty because of the growing number of elderly people in the general population. The Longevity-Associated Variant (LAV) of the human BPIFB4 gene was found to correct endothelial dysfunction, one of the mechanisms underlying frailty, in aging mice whereas the RV-BPIFB4 variant induced opposite effects. Thus, we newly hypothesize that, besides being associated with life expectancy, BPIFB4 polymorphisms can predict frailty.Aim and Results: Here we investigated if the BPIFB4 haplotypes, LAV, wild-type (WT) and RV, differentially associate with frailty in a cohort of 237 elderly subjects from Calabria region in Southern Italy. Moreover, we studied the effect of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the progression of frailty in aging mice. We found an inverse correlation of the homozygous LAV-BPIFB4 haplotype with frailty in elderly subjects. Conversely, carriers of the RV-BPIFB4 haplotype showed an increase in the frailty status and risk of death. Moreover, in old mice, LAV-BPIFB4 gene transfer delayed frailty progression.Conclusions: These data indicate that specific BPIFB4 haplotypes could represent useful genetic markers of frailty. In addition, horizontal transfer of a healthy gene variant can attenuate frailty in aging organisms.

Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-β and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The application of chondrogenic gene sequences to human bone marrow-derived mesenchymal stromal cells (hMSCs) is an attractive strategy to activate the reparative activities of these cells as a means to enhance the processes of cartilage repair using indirect cell transplantation procedures that may improve the repopulation of cartilage lesions. In the present study, we examined the feasibility of co-delivering the highly competent transforming growth factor beta (TGF-β) with the insulin-like growth factor I (IGF-I) in hMSCs via recombinant adeno-associated virus (rAAV) vector-mediated gene transfer prior to implantation in a human model of osteochondral defect (OCD) ex vivo that provides a microenvironment similar to that of focal cartilage lesions. The successful co-overexpression of rAAV TGF-β/IGF-I in implanted hMSCs promoted the durable remodeling of tissue injury in human OCDs over a prolonged period of time (21 days) relative to individual gene transfer and the control (reporter lacZ gene) treatment, with enhanced levels of cell proliferation and matrix deposition (proteoglycans, type-II collagen) both in the lesions and at a distance, while hypertrophic, osteogenic, and catabolic processes could be advantageously delayed. These findings demonstrate the value of indirect, progenitor cell-based combined rAAV gene therapy to treat human focal cartilage defects in a natural environment as a basis for future clinical applications.

Gene therapy prevents hepatic tumor initiation in murine glycogen storage disease type Ia at the tumor-developing stage.

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long-term complication of glycogen storage disease type-Ia (GSD-Ia), which is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in gluconeogenesis. Currently, there is no therapy to address HCA/HCC in GSD-Ia. We have previously shown that a recombinant adeno-associated virus (rAAV) vector-mediated G6PC gene transfer to 2-week-old G6pc-/- mice prevents HCA development. However, it remains unclear whether G6PC gene transfer at the tumor developing stage of GSD-Ia can prevent tumor initiation or abrogate the pre-existing tumors. Using liver-specific G6pc-knockout (L-G6pc-/-) mice that develop HCA/HCC, we now show that treating the mice at the tumor-developing stage with rAAV-G6PC restores hepatic G6Pase-α expression, normalizes glucose homeostasis, and prevents de novo HCA/HCC development. The rAAV-G6PC treatment also normalizes defective hepatic autophagy and corrects metabolic abnormalities in the nontumor liver tissues of both tumor-free and tumor-bearing mice. However, gene therapy cannot restore G6Pase-α expression in the HCA/HCC lesions and fails to abrogate any pre-existing tumors. We show that the expression of 11 β-hydroxysteroid dehydrogenase type-1 that mediates local glucocorticoid activation is downregulated in HCA/HCC lesions, leading to impairment in glucocorticoid signaling critical for gluconeogenesis activation. This suggests that local glucocorticoid action downregulation in the HCA/HCC lesions may suppress gene therapy mediated G6Pase-α restoration. Collectively, our data show that rAAV-mediated gene therapy can prevent de novo HCA/HCC development in L-G6pc-/- mice at the tumor developing stage, but it cannot reduce any pre-existing tumor burden.

Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector-mediated intrauterine gene transfer in early-gestation fetal macaques.

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 1012 vector genomes (vgs) of AAV5-hFIX (n = 5; 0.45 × 1013 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6–6.8% over 19–51 mo. The next cohort received 0.2–1 × 1013 vg boluses. AAV5-hFX animals (n = 3; 3.57 × 1013 vg/kg) expressed <1% at birth and 9.4–27.9% up to 42 mo. AAV8-hFIX recipients (n = 3; 2.56 × 1013 vg/kg) established 4.2–41.3% expression perinatally and 9.8–25.3% over 46 mo. Expression with AAV8-hFX (n = 6, 3.12 × 1013 vg/kg) increased from <1% perinatally to 9.8–13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 × 1011 vg/kg AAV5 resulting in 2.4–13.2% expression and demonstrating acquired tolerance. Linear amplification–mediated-PCR analysis demonstrated random integration of 57–88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.—Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector–mediated intrauterine gene transfer in early-gestation fetal macaques.

Post-injury Nose-to-Brain Delivery of Activin A and SerpinB2 Reduces Brain Damage in a Mouse Stroke Model.

Synaptic NMDA receptors activating nuclear calcium-driven adaptogenomics control a potent body-own neuroprotective mechanism, referred to as acquired neuroprotection. Viral vector-mediated gene transfer in conjunction with stereotactic surgery has previously demonstrated the proficiency of several nuclear calcium-regulated genes to protect invivo against braindamage caused by toxic extrasynaptic NMDA receptor signaling following seizures or stroke. Here we used noninvasive nose-to-brain administration of Activin A and SerpinB2, two secreted nuclear calcium-regulated neuroprotectants, for post-injury treatment of brain damage following middle cerebral artery occlusion (MCAO) in C57BL/6N mice. The observed reduction of the infarct volume was comparable to the protection obtained by intracerebroventricular injection of recombinant Activin A or SerpinB2 or by stereotactic delivery 3weeks prior to the injury of a recombinant adeno-associated virus containing an expression cassette for the potent neuroprotective transcription factor Npas4. These results establish post-injury, nose-to-brain delivery of Activin A and SerpinB2 as effective and possibly clinically applicable treatments of acute and chronic neurodegenerative conditions.

Adeno-associated viral vector-mediated ATPase gene transfer alleviates asthmatic airway inflammation in OVA-sensitized mice model

Abstract Bronchial asthma is a chronic inflammatory disorder of the airway characterized by airway hyperresponsiveness (AHR), pulmonary eosinophilia, elevated serum IgE, and dysregulated T helper (Th) 2 cytokines. It is becoming urgent to identify target molecules for the preventive and therapeutic purpose. ATP accumulation in bronchoalveolar lavage fluid (BALF) of asthmatic subjects indicates that extracellular ATP and the downstream responses are involved. ATP is tightly regulated by CD39, a nucleoside triphosphate diphosphohydrolase (ATPase) encoded by entpd1. Upregulated BALF ATP levels and decreased CD39 RNA were accompanied with asthmatic cardinal features in ovalbumin (OVA)-sensitized mice. Recombinant adeno-associated virus (rAAV) has been developed to carry exogenous genes with safe and low toxic therapeutic potential. The different serotypes and tropisms of AAVs benefit tissue specific gene targeting. This study aims to determine whether lung-targeting rAAV carrying CD39 (AAV/CD39) is able to reduce extracellular ATP in BALF and inhibit the OVA-induced allergic inflammation in an asthmatic murine model. rAAV vectors were administrated into the lungs of mice 3 days before the first OVA exposure. The data indicate that ATP levels in BALF were downregulated accompanied with rescued CD39 expression. The administration of AAV/CD39 was able to suppress AHR, pulmonary eosinophilia, and the production of IL-5 and IL-13 from OVA-activated mediastinal lymph nodes (MLNs) cells. IL-33 in BALF was also reduced. These data indicate that the attenuation of asthmatic airway inflammation may affect innate lymphoid cells (ILC) 2 activation. Thus, ATP level could be a potential target for asthma gene therapy.

Stereotaxic Surgery and Viral Delivery of Zinc-Finger Epigenetic Editing Tools in Rodent Brain.

Delivery of engineered zinc-finger proteins (ZFPs) for targeted epigenetic remodeling in rodent brain can be facilitated by the use of viral vector-mediated gene transfer coupled with stereotaxic surgery techniques. Here we describe the surgical protocol utilized by our group which is optimized for herpes simplex virus (HSV) delivery into mouse brain. The protocol outlined herein could be applied for delivery of adeno-associated viruses (AAV) or lentiviruses in both mice and rats. This method allows for the viral expression of engineered DNA-binding factors, particularly engineered ZFPs, and subsequent epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of in vivo neuroepigenetic editing with synthetic ZFP constructs.

Viral Expression of Epigenome Editing Tools in Rodent Brain Using Stereotaxic Surgery Techniques.

Delivery of molecular tools for targeted epigenome editing in rodent brain can be facilitated by the use of viral vector-mediated gene transfer coupled with stereotaxic surgery techniques. Here, we describe the surgical protocol utilized by our group, which is optimized for herpes simplex virus (HSV)-mediated delivery into mouse brain. The protocol outlined herein could also be applied for delivery of adeno-associated viruses (AAV) or lentiviruses in both mice and rats. This method allows for efficient viral transgene expression and subsequent epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of in vivo neuroepigenome editing.

Rescue of GSDIII Phenotype with Gene Transfer Requires Liver- and Muscle-Targeted GDE Expression.

Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liverexpression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the factor VIII (FVIII) coagulation protein. Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma-derived FVIII. We have made an adeno-associated virus 5 vector containing a B domain-deleted (BDD) FVIII gene (BMN 270) with a liver-specific promoter. BMN 270 injected into hemophilic mice resulted in a dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested, complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by BMN 270 or following exogenous administration of recombinant BDD FVIII. No evidence of liver dysfunction or hepatocyte endoplasmic reticulum stress was observed. Comparable doses in primates produced similar levels of circulating FVIII. These preclinical data support evaluation of BMN 270 in hemophilia A patients.

Duplex Quantitative Polymerase Chain Reaction Assay for Detection of Adenoviral and Lentiviral Vectors

The ability to detect and quantify viral vector sequences from a variety of clinical sample types is crucial to performing biosafety risk assessments. Viral vector-mediated gene transfer studies ar...

Spinal nociceptive circuit analysis with recombinant adeno-associated viruses: the impact of serotypes and promoters.

Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a >20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.

AAV-Mediated Gene Supplementation Therapy in Achromatopsia Type 2: Preclinical Data on Therapeutic Time Window and Long-Term Effects.

Achromatopsia type 2 (ACHM2) is a severe, inherited eye disease caused by mutations in the CNGA3 gene encoding the α subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Patients suffer from strongly impaired daylight vision, photophobia, nystagmus, and lack of color discrimination. We have previously shown in the Cnga3 knockout (KO) mouse model of ACHM2 that gene supplementation therapy is effective in rescuing cone function and morphology and delaying cone degeneration. In our preclinical approach, we use recombinant adeno-associated virus (AAV) vector-mediated gene transfer to express the murine Cnga3 gene under control of the mouse blue opsin promoter. Here, we provide novel data on the efficiency and permanence of such gene supplementation therapy in Cnga3 KO mice. Specifically, we compare the influence of two different AAV vector capsids, AAV2/5 (Y719F) and AAV2/8 (Y733F), on restoration of cone function, and assess the effect of age at time of treatment on the long-term outcome. The evaluation included in vivo analysis of retinal function using electroretinography (ERG) and immunohistochemical analysis of vector-driven Cnga3 transgene expression. We found that both vector capsid serotypes led to a comparable rescue of cone function over the observation period between 4 weeks and 3 months post treatment. In addition, a clear therapeutic effect was present in mice treated at 2 weeks of age as well as in mice treated at 3 months of age at the first assessment at 4 weeks after treatment. Importantly, the effect extended in both cases over the entire observation period of 12 months post treatment. However, the average ERG amplitude levels differed between the two groups, suggesting a role of the absolute age, or possibly, the associated state of the degeneration, on the achievable outcome. In summary, we found that the therapeutic time window of opportunity for AAV-mediated Cnga3 gene supplementation therapy in the Cnga3 KO mouse model extends at least to an age of 3 months, but is presumably limited by the condition, number and topographical distribution of remaining cones at the time of treatment. No impact of the choice of capsid on the therapeutic success was detected.

Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken β-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.