A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes.

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently in vivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

Antibody-based protection against respiratory syncytial virus in mice and their offspring through vectored immunoprophylaxis.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections, with potential lower respiratory tract infections, which can be particularly problematic in infants and the elderly. There are no approved vaccines for RSV. The current standard of care for high-risk individuals is monthly administration of palivizumab, a humanized murine monoclonal antibody (mAb) targeting the RSV fusion protein. Adeno-associated virus (AAV)-mediated expression of mAbs has previously led to sustained expression of therapeutic concentrations of mAbs in several animal models, representing an alternative to repetitive passive administration. Intramuscular (IM) administration of AAV6.2FF expressing RSV antibodies, palivizumab or hRSV90, resulted in high concentrations of human (h)IgG1 mAbs in the serum and at various mucosal surfaces, while intranasal administration limited hIgG expression to the respiratory tract. IM administration of AAV6.2FF-hRSV90 or AAV6.2FF-palivizumab in a murine model provided sterilizing immunity against challenge with RSV A2. Evidence of maternal passive transfer of vectorized hRSV90 was detected in both murine and ovine models, with circulating mAbs providing sterilizing immunity in mouse progeny. Finally, addition of a "kill switch" comprised of LoxP sites flanking the mAb genes resulted in diminished serum hIgG after AAV-DJ-mediated delivery of Cre recombinase to the same muscle group that was originally transduced with the AAV-mAb vector. The ability of this AAV-mAb system to mediate robust, sustained mAb expression for maternal transfer to progeny in murine and ovine models emphasizes the potential of this platform for use as an alternative prophylactic vaccine for protection against neonatal infections, particularly in high-risk infants.

AAV-mediated delivery of actoxumab and bezlotoxumab results in serum and mucosal antibody concentrations that provide protection from C. difficile toxin challenge.

Clostridium difficile is the leading cause of antibiotic-associated nosocomial diarrhea in the developed world. When the host-associated colon microbiome is disrupted by the ingestion of antibiotics, C. difficile spores can germinate, resulting in infection. C. difficile secretes enterotoxin A (TcdA) and cytotoxin B (TcdB) that are responsible for disease pathology. Treatment options are limited as the bacterium demonstrates resistance to many antibiotics, and even with antibacterial therapies, recurrences of C. difficile are common. Actotoxumab and bezlotoxumab are human monoclonal antibodies that bind and neutralize TcdA and TcdB, respectively. In 2016, the US food and drug administration (FDA) approved bezlotoxumab for use in the prevention of C. difficile infection recurrence. To ensure the long-term expression of antibodies, gene therapy can be used. Here, adeno-associated virus (AAV)6.2FF, a novel triple mutant of AAV6, was engineered to express either actotoxumab or bezlotoxumab in mice and hamsters. Both antibodies expressed at greater than 90 µg/mL in the serum and were detected at mucosal surfaces in both models. Hundred percent of mice given AAV6.2FF-actoxumab survived a lethal dose of TcdA. This proof of concept study demonstrates that AAV-mediated expression of C. difficile toxin antibodies is a viable approach for the prevention of recurrent C. difficile infections.

Engineered AAV8 capsid acquires heparin and AVB sepharose binding capacity but has altered in vivo transduction efficiency.

Naturally occurring adeno-associated virus (AAV) serotypes that bind to ligands such as AVB sepharose or heparin can be purified by affinity chromatography, which is a more efficient and scalable method than gradient ultracentrifugation. Wild-type AAV8 does not bind effectively to either of these molecules, which constitutes a barrier to using this vector when a high throughput design is required. Previously, AAV8 was engineered to contain a SPAKFA amino acid sequence to facilitate purification using AVB sepharose resin; however, in vivo studies were not conducted to examine whether these capsid mutations altered the transduction profile. To address this gap in knowledge, a mutant AAV8 capsid was engineered to bind to AVB sepharose and heparan sulfate (AAV8-AVB-HS), which efficiently bound to both affinity columns, resulting in elution yields of >80% of the total vector loaded compared to <5% for wild-type AAV8. However, in vivo comparison by intramuscular, intravenous, and intraperitoneal vector administration demonstrated a significant decrease in AAV8-AVB-HS transduction efficiency without alteration of the transduction profile. Therefore, although it is possible to engineer AAV capsids to bind various affinity ligands, the consequences associated with mutating surface exposed residues have the potential to negatively impact other vector characteristics including in vivo potency and production yield. This study demonstrates the importance of evaluating all aspects of vector performance when engineering AAV capsids.

AAV Vectors Pseudotyped with Capsids from Porcine and Bovine Species Mediate In Vitro and In Vivo Gene Delivery.

Adeno-associated virus (AAV) vectors are among the most widely used delivery vehicles for in vivo gene therapy as they mediate robust and sustained transgene expression with limited toxicity. However, a significant impediment to the broad clinical success of AAV-based therapies is the widespread presence of pre-existing humoral immunity to AAVs in the human population. This immunity arises from the circulation of non-pathogenic endemic human AAV serotypes. One possible solution is to use non-human AAV capsids to pseudotype transgene-containing AAV vector genomes of interest. Due to the low probability of human exposure to animal AAVs, pre-existing immunity to animal-derived AAV capsids should be low. Here, we characterize two novel AAV capsid sequences: one derived from porcine colon tissue and the other from a caprine adenovirus stock. Both AAV capsids proved to be effective transducers of HeLa and HEK293T cells in vitro. In vivo, both capsids were able to transduce the murine nose, lung, and liver after either intranasal or intraperitoneal administration. In addition, we demonstrate that the porcine AAV capsid likely arose from multiple recombination events involving human- and animal-derived AAV sequences. We hypothesize that recurrent recombination events with similar and distantly related AAV sequences represent an effective mechanism for enhancing the fitness of wildtype AAV populations.

Adeno-associated virus mediated expression of monoclonal antibody MR191 protects mice against Marburg virus and provides long-term expression in sheep.

Vectored monoclonal antibody (mAb) expression mediated by adeno-associated virus (AAV) gene delivery leads to sustained therapeutic mAb expression and protection against a wide range of infectious diseases in both small and large animal models, including nonhuman primates. Using our rationally engineered AAV6 triple mutant capsid, termed AAV6.2FF, we demonstrate rapid and robust expression of two potent human antibodies against Marburg virus, MR78 and MR191, following intramuscular (IM) administration. IM injection of mice with 1 × 1011 vector genomes (vg) of AAV6.2FF-MR78 and AAV6.2FF-MR191 resulted in serum concentrations of approximately 141 µg/mL and 195 µg/mL of human IgG, respectively, within the first four weeks. Mice receiving 1 × 1011 vg (high) and 1 × 1010 vg (medium) doses of AAV6.2FF-MR191 were completely protected against lethal Marburg virus challenge. No sex-based differences in serum human IgG concentrations were observed; however, administering the AAV-mAb over multiple injection sites significantly increased serum human IgG concentrations. IM administration of three two-week-old lambs with 5 × 1012 vg/kg of AAV6.2FF-MR191 resulted in serum human IgG expression that was sustained for more than 460 days, concomitant with low levels of anti-capsid and anti-drug antibodies. AAV-mAb expression is a viable method for prolonging the therapeutic effect of recombinant mAbs and represents a potential alternative "vaccine" strategy for those with compromised immune systems or in possible outbreak response scenarios.

AAV-monoclonal antibody expression protects mice from Ebola virus without impeding the endogenous antibody response to heterologous challenge.

Filoviruses cause severe hemorrhagic fever with case fatality rates as high as 90%. Filovirus-specific monoclonal antibodies (mAbs) confer protection in nonhuman primates as late as 5 days after challenge, and FDA-approved mAbs REGN-EB3 and mAb114 have demonstrated efficacy against Ebola virus (EBOV) infection in humans. Vectorized antibody expression mediated by adeno-associated virus (AAV) can generate protective and sustained concentrations of therapeutic mAbs in animal models for a variety of infectious diseases, including EBOV. Here we demonstrate that AAV6.2FF-mediated expression of murine IgG2a EBOV mAbs, 2G4 and 5D2, protects from mouse-adapted (MA)-EBOV infection with none of the surviving mice developing anti-VP40 antibodies above background. Protective serum concentrations of AAV6.2FF-2G4/AAV6.2FF-5D2 did not alter endogenous antibody responses to heterologous virus infection. AAV-mediated expression of EBOV mAbs 100 and 114, and pan-ebolavirus mAbs, FVM04, ADI-15878, and CA45, as human IgG1 antibodies conferred protection against MA-EBOV at low serum concentrations, with minimum protective serum levels as low as 2 µg/mL. Vectorized expression of murine IgG2a or human IgG1 mAbs led to sustained expression in the serum of mice for >400 days or for the lifetime of the animal, respectively. AAV6.2FF-mediated mAb expression offers an alternative to recombinant antibody administration in scenarios where long-term protection is preferable to passive immunization.

Safety and Tolerability of the Adeno-Associated Virus Vector, AAV6.2FF, Expressing a Monoclonal Antibody in Murine and Ovine Animal Models.

Adeno-associated virus (AAV) vector mediated expression of therapeutic monoclonal antibodies is an alternative strategy to traditional vaccination to generate immunity in immunosuppressed or immunosenescent individuals. In this study, we vectorized a human monoclonal antibody (31C2) directed against the spike protein of SARS-CoV-2 and determined the safety profile of this AAV vector in mice and sheep as a large animal model. In both studies, plasma biochemical parameters and hematology were comparable to untreated controls. Except for mild myositis at the site of injection, none of the major organs revealed any signs of toxicity. AAV-mediated human IgG expression increased steadily throughout the 28-day study in sheep, resulting in peak concentrations of 21.4-46.7 µg/ mL, demonstrating practical scale up from rodent to large animal models. This alternative approach to immunity is worth further exploration after this demonstration of safety, tolerability, and scalability in a large animal model.

AAV Vectored Immunoprophylaxis for Filovirus Infections.

Filoviruses are among the deadliest infectious agents known to man, causing severe hemorrhagic fever, with up to 90% fatality rates. The 2014 Ebola outbreak in West Africa resulted in over 28,000 infections, demonstrating the large-scale human health and economic impact generated by filoviruses. Zaire ebolavirus is responsible for the greatest number of deaths to date and consequently there is now an approved vaccine, Ervebo, while other filovirus species have similar epidemic potential and remain without effective vaccines. Recent clinical success of REGN-EB3 and mAb-114 monoclonal antibody (mAb)-based therapies supports further investigation of this treatment approach for other filoviruses. While efficacious, protection from passive mAb therapies is short-lived, requiring repeat dosing to maintain therapeutic concentrations. An alternative strategy is vectored immunoprophylaxis (VIP), which utilizes an adeno-associated virus (AAV) vector to generate sustained expression of selected mAbs directly in vivo. This approach takes advantage of validated mAb development and enables vectorization of the top candidates to provide long-term immunity. In this review, we summarize the history of filovirus outbreaks, mAb-based therapeutics, and highlight promising AAV vectorized approaches to providing immunity against filoviruses where vaccines are not yet available.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Optimized Pre-Clinical Grade Production of Two Novel Lentiviral Vector Pseudotypes for Lung Gene Delivery.

Lung gene therapy requires efficient transduction of slow-replicating epithelia and stable expression of delivered transgenes in the respiratory tract. Lentiviral (LV) vectors have the ideal coding, expression, and transducing capacity required for gene therapy. A modified envelope glycoprotein from the Jaagsiekte Sheep Retrovirus, termed Jenv, is well suited for LV-mediated lung gene therapy due to its inherent lung tropism. Here, two novel Jenv-pseudotyped LVs that effectively transduce lung tissue and yield titers similar to the gold standard, vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped LVs, were generated. As the concentration efficiency of LVs was found to depend on envelope pseudotype, a large-scale production method tailored for Jenv-pseudotyped LVs was developed and the most appropriate method of concentration was determined. In contrast to VSVg and Ebola virus glycoprotein-pseudotyped LVs, ultracentrifugation through a sucrose cushion drastically reduced the yield of Jenv LVs, whereas polyethylene glycol precipitation and tangential flow filtration (TFF) proved to be more suitable methods for concentrating Jenv LVs. Importantly, pressure during TFF was found to be crucial for increasing LV recovery. Finally, a unique mouse model was developed to test the suitability of these novel Jenv-pseudotyped LVs for use in lung gene therapy applications.

The U3 and Env Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Both Contribute to Tissue Tropism.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are small-ruminant betaretroviruses that share high nucleotide and amino acid identity, utilize the same cellular receptor, hyaluronoglucosaminidase 2 (Hyal2) for entry, and transform tissues with their envelope (Env) glycoprotein; yet, they target discrete regions of the respiratory tract-the lung and nose, respectively. This distinct tissue selectivity makes them ideal tools with which to study the pathogenesis of betaretroviruses. To uncover the genetic determinants of tropism, we constructed JSRV-ENTV chimeric viruses and produced lentivectors pseudotyped with the Env proteins from JSRV (Jenv) and ENTV (Eenv). Through the transduction and infection of lung and nasal turbinate tissue slices, we observed that Hyal2 expression levels strongly influence ENTV entry, but that the long terminal repeat (LTR) promoters of these viruses are likely responsible for tissue-specificity. Furthermore, we show evidence of ENTV Env expression in chondrocytes within ENTV-infected nasal turbinate tissue, where Hyal2 is highly expressed. Our work suggests that the unique tissue tropism of JSRV and ENTV stems from the combined effort of the envelope glycoprotein-receptor interactions and the LTR and provides new insight into the pathogenesis of ENTV.

AAV-Mediated Gene Delivery to the Lung.

Adeno-associated virus (AAV) vectors are an efficient method of gene delivery to various tissues including the lung. Mouse models are often used as a preliminary preclinical model in order to advance AAV lung gene therapy vectors. In this chapter we describe an AAV purification protocol using heparin affinity chromatography as well as an intranasal and intratracheal method of delivering AAV vectors to the lungs of mice.

A Novel Triple-Mutant AAV6 Capsid Induces Rapid and Potent Transgene Expression in the Muscle and Respiratory Tract of Mice.

Gene therapy for the treatment of genetic disorders has demonstrated considerable therapeutic success in clinical trials. Among the most effective and commonly used gene delivery vectors are those based on adeno-associated virus (AAV). Despite these advances in clinical gene therapy, further improvements in AAV vector properties such as rapid intracellular processing and transgene expression, targeted transduction of therapeutically relevant cell types, and longevity of transgene expression, will render extension of such successes to many other human diseases. Engineering of AAV capsids continues to evolve the specificity and efficiency of AAV-mediated gene transfer. Here, we describe a triple AAV6 mutant, termed AAV6.2FF, containing F129L, Y445F, and Y731F mutations. AAV6.2FF yielded 10-fold greater transgene expression in lung than AAV6 after 21 days. Additionally, this novel capsid demonstrated 101-fold and 49-fold increased transgene expression in the muscle and lungs, respectively, 24 hr post vector delivery when compared with the parental AAV6. Furthermore, AAV6.2FF retains heparin sulfate binding capacity and displays a 10-fold increase in resistance to pooled immunoglobulin neutralization in vitro. The rapid and potent expression mediated by AAV6.2FF is ideally suited to applications such as vectored immunoprophylaxis, in which rapid transgene expression is vital for use during an outbreak response scenario.

Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

AAV vector distribution in the mouse respiratory tract following four different methods of administration.

BACKGROUND: Targeted delivery of gene therapy vectors to the mouse respiratory tract is often performed via intranasal or intratracheal administration; however, there can be a great deal of variability between these methods, which could potentially influence experimental results. Improving the accuracy and precision of lung delivery will not only reduce the number of animals required to detect statistically significant differences, but may reduce the variability of studies from different laboratories. RESULTS: Here we evaluated three different methods of adeno-associated virus (AAV) vector administration to the respiratory tract in mice (intranasal, intubation, and intratracheal injection) and discuss the advantages, challenges, and shortcomings of each. We also present a modified-intranasal delivery technique that is superior to passive administration of vector into the nares of anesthetized supine animals. Transgene expression was consistently visible in the nasal cavity, trachea, and proximal to middle aspect of all lung lobes for all four methods, whereas transgene expression was consistently observed in the most distal aspect of lung lobes only with the intubation and intratracheal injection techniques. AAV vector genome copy numbers in the lung were approximately four-fold lower in mice that received vector via intranasal administration in comparison to the other three methods of vector delivery. The modified intranasal, intubation and intratracheal injection methods of vector administration did not yield statistical differences in AAV vector genome copy numbers in the lung. With regard to reproducibility of vector distribution within and between animals, the modified-intranasal technique was superior. CONCLUSION: Our results show that mode of AAV vector administration to the murine respiratory tract should be selected based on desired target site and skill of the researcher, and that appropriate technique selection may greatly influence experimental outcomes.