Inhalation of SP-101 Followed by Inhaled Doxorubicin Results in Robust and Durable hCFTRΔR Transgene Expression in the Airways of Wild-Type and Cystic Fibrosis Ferrets.

Cystic fibrosis (CF) is a serious genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Approved small molecule therapies benefit the majority of people with CF (pwCF), but unfortunately not all. Gene addition offers a mutation agnostic treatment option for all pwCF. SP-101 is an adeno-associated virus gene therapy vector (AAV2.5T) that has been optimized for efficient human airway cell transduction, and that contains a functional and regulated shortened human CFTR minigene (hCFTRΔR) with a small synthetic promoter/enhancer. To understand SP-101 airway distribution, activity, and the associated immune response, in vivo studies were performed in wild-type and CF ferrets. After single dose inhaled delivery of SP-101, followed by single dose inhaled doxorubicin (an AAV transduction augmenter) or saline, SP-101 vector genomes were detected throughout the respiratory tract. hCFTRΔR mRNA expression was highest in ferrets also receiving doxorubicin and persisted for the duration of the study (13 weeks). Pre-existing mucus in the CF ferrets did not present a barrier to effective transduction. Binding and neutralizing antibodies to the AAV2.5T capsid were observed regardless of doxorubicin exposure. Only a portion of ferrets exhibited a weak T-cell response to AAV2.5T and no T-cell response was seen against hCFTRΔR. These data strongly support the continued development of inhaled SP-101, followed by inhaled doxorubicin, for the treatment of CF.

SP-101, A Novel Adeno-Associated Virus Gene Therapy for the Treatment of Cystic Fibrosis, Mediates Functional Correction of Primary Human Airway Epithelia From Donors with Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Although CF affects multiple organs, lung disease is the main cause of morbidity and mortality, and gene therapy is expected to provide a mutation-agnostic option for treatment. SP-101 is a recombinant adeno-associated virus (AAV) gene therapy vector carrying a human CFTR minigene, hCFTRΔR, and is being investigated as an inhalation treatment for people with CF. To further understand SP-101 activity, in vitro studies were performed in human airway epithelia (HAE) derived from multiple CF and non-CF donors. SP-101 restored CFTR-mediated chloride conductance, measured via Ussing chamber assay, at a multiplicity of infection (MOI) as low as 5E2 in the presence of doxorubicin, a small molecule known to augment AAV transduction. Functional correction of CF HAE increased with increasing MOI and doxorubicin concentration and correlated with increasing cell-associated vector genomes and hCFTRΔR mRNA expression. Tropism studies using a fluorescent reporter vector and single-cell mRNA sequencing of SP-101-mediated hCFTRΔR mRNA demonstrated broad expression in all cell types after apical transduction, including secretory, ciliated, and basal cells. In summary, SP-101, particularly in combination with doxorubicin, shows promise for a novel CF treatment strategy and strongly supports continued development.

Durable transgene expression and efficient re-administration after rAAV2.5T-mediated fCFTRΔR gene delivery to adult ferret lungs.

The dosing interval for effective recombinant adeno-associated virus (rAAV)-mediated gene therapy of cystic fibrosis lung disease remains unknown. Here, we assessed the durability of rAAV2.5T-fCFTRΔR-mediated transgene expression and neutralizing antibody (NAb) responses in lungs of adult wild-type ferrets. Within the first 3 months following rAAV2.5T-fCFTRΔR delivery to the lung, CFTRΔR transgene expression declined ∼5.6-fold and then remained stable to 5 months at ∼26% the level of endogenous CFTR. rAAV NAbs in the plasma and bronchoalveolar lavage fluid (BALF) peaked at 21 days, coinciding with peak ELISpot T cell responses to AAV capsid peptides, after which both responses declined and remained stable at 4-5 months post dosing. Administration of reporter vector rAAV2.5T-gLuc (gaussia luciferase) at 5 months following rAAV2.5T-fCFTRΔR dosing gave rise to similar levels of gLuc expression in the BALF as observed in age-matched reporter-only controls, demonstrating that residual BALF NAbs were functionally insignificant. Notably, the second vector administration led to a 2.6-fold greater ELISpot T cell response and ∼2.3-fold decline in fCFTRΔR mRNA and vector genomes derived from the initial rAAV2.5T-fCFTRΔR administration, suggesting selective destruction of transduced cells from the first vector dose. These findings provide insights into humoral and cellular immune response to rAAV that may be useful for optimizing gene therapy to the cystic fibrosis lung.

Restoring Ciliary Function: Gene Therapeutics for Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by defects in motile cilia, which play an important role in several organ systems. Lung disease is a hallmark of PCD, given the essential role of cilia in airway surface defense. Diagnosis of PCD is complicated due to its reliance on complex tests that are not utilized by every clinic and also its phenotypic overlap with several other respiratory diseases. Nonetheless, PCD is increasingly being recognized as more common than once thought. The disease is genetically complex, with several genes reported to be associated with PCD. There is no cure for PCD, but gene therapy remains a promising therapeutic strategy. In this review, we provide an overview of the clinical symptoms, diagnosis, genetics, and current treatment regimens for PCD. We also describe PCD model systems and discuss the therapeutic potential of different gene therapeutics for targeting the intended cellular target, the ciliated cells of the airway.

Immunosuppression reduces rAAV2.5T neutralizing antibodies that limit efficacy following repeat dosing to ferret lungs.

The efficacy of redosing the recombinant adeno-associated virus (rAAV) vector rAAV2.5T to ferret lung is limited by AAV neutralizing antibody (NAb) responses. While immunosuppression strategies have allowed for systemic rAAV repeat dosing, their utility for rAAV lung-directed gene therapy is largely unexplored. To this end, we evaluated two immunosuppression (IS) strategies to improve repeat dosing of rAAV2.5T to ferret lungs: (1) a combination of three IS drugs (Tri-IS) with broad coverage against cellular and humoral responses (methylprednisolone [MP], azathioprine, and cyclosporine) and (2) MP alone, which is typically used in systemic rAAV applications. Repeat dosing utilized AAV2.5T-SP183-fCFTRΔR (recombinant ferret CFTR transgene), followed 28 days later by AAV2.5T-SP183-gLuc (for quantification of transgene expression). Both the Tri-IS and MP strategies significantly improved transgene expression following repeat dosing and reduced AAV2.5T NAb responses in the bronchioalveolar lavage fluid (BALF) and plasma, while AAV2.5T binding antibody subtypes and cellular immune responses by ELISpot were largely unchanged by IS. One exception was the reduction in plasma AAV2.5T binding immunoglobulin G (IgG) in both IS groups. Only the Tri-IS strategy significantly suppressed splenocyte expression of IFNA (interferon α [IFN-α]) and IL4. Our studies suggest that IS strategies may be useful in clinical application of rAAV targeting lung genetic diseases such as cystic fibrosis.

Gene Therapeutics for Surfactant Dysfunction Disorders: Targeting the Alveolar Type 2 Epithelial Cell.

Genetic disorders of surfactant dysfunction result in significant morbidity and mortality, among infants, children, and adults. Available medical interventions are limited, nonspecific, and generally ineffective. As such, the need for effective therapies remains. Pathogenic variants in the SFTPB, SFTPC, and ABCA3 genes, each of which encode proteins essential for proper pulmonary surfactant production and function, result in interstitial lung disease in infants, children, and adults, and lead to morbidity and early mortality. Expression of these genes is predominantly limited to the alveolar type 2 (AT2) epithelial cells present in the distal airspaces of the lungs, thus providing an unequivocal cellular origin of disease pathogenesis. While several treatment strategies are under development, a gene-based therapeutic holds great promise as a definitive therapy. Importantly for clinical translation, the genes associated with surfactant dysfunction are both well characterized and amenable to a gene-therapeutic-based strategy. This review focuses on the pathophysiology associated with these genetic disorders of surfactant dysfunction, and also provides an overview of the current state of gene-based therapeutics designed to target and transduce the AT2 cells.

Adeno-Associated Virus Vector-Mediated Expression of Antirespiratory Syncytial Virus Antibody Prevents Infection in Mouse Airways.

Infants and older adults are especially vulnerable to infection by respiratory syncytial virus (RSV), which can cause significant illness and irreparable damage to the lower respiratory tract and for which an effective vaccine is not readily available. Palivizumab, a recombinant monoclonal antibody (mAb), is an approved therapeutic for RSV infection for use in high-risk infants only. Due to several logistical issues, including cost of goods and scale-up limitations, palivizumab is not approved for other populations that are vulnerable to severe RSV infections, such as older adults. In this study, we demonstrate that intranasal delivery of adeno-associated virus serotype 9 (AAV9) vector expressing palivizumab or motavizumab, a second-generation version of palivizumab, significantly reduced the viral load in the lungs of the BALB/c mouse model of RSV infection. Notably, we demonstrate that AAV9 vector-mediated prophylaxis against RSV was effective despite the presence of serum-circulating neutralizing AAV9 antibodies. These findings substantiate the feasibility of repeatedly administering AAV9 vector to the airway for seasonal prophylaxis against RSV, thereby expanding the application of vectored delivery of mAbs as an effective prophylaxis strategy against various airborne viruses.

Adeno-associated virus-mediated expression of human butyrylcholinesterase to treat organophosphate poisoning.

Rare diseases defined by genetic mutations are classic targets for gene therapy. More recently, researchers expanded the use of gene therapy in non-clinical studies to infectious diseases through the delivery of vectorized antibodies to well-defined antigens. Here, we further extend the utility of gene therapy beyond the "accepted" indications to include organophosphate poisoning. There are no approved preventives for the multi-organ damage resulting from acute or chronic exposure to organophosphates. We show that a single intramuscular injection of adeno-associated virus vector produces peak expression (~0.5 mg/ml) of active human butyrylcholinesterase (hBChE) in mice serum within 3-4 weeks post-treatment. This expression is sustained for up to 140 days post-injection with no silencing. Sustained expression of hBChE provided dose-dependent protection against VX in male and female mice despite detectable antibodies to hBChE in some mice, thereby demonstrating that expression of hBChE in vivo in mouse muscle is an effective prophylactic against organophosphate poisoning.

Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

Intrathecal Viral Vector Delivery of Trastuzumab Prevents or Inhibits Tumor Growth of Human HER2-Positive Xenografts in Mice.

Breast cancer brain metastases are a deadly sequela of primary breast tumors that overexpress human epidermal growth factor receptor 2 (HER2); median survival for patients with these tumors is 10 to 13 months from the time of diagnosis. Current treatments for HER2-positive breast cancer brain metastases are invasive, toxic, and largely ineffective. Here, we have developed an adeno-associated virus serotype 9 (AAV9) vector to express the anti-HER2 monoclonal antibody trastuzumab (Herceptin) in vivo A single prophylactic intrathecal administration of AAV9.trastuzumab vector in a novel orthotopic Rag1-/- murine xenograft model of HER2-positive breast cancer brain metastases significantly increased median survival, attenuated brain tumor growth, and preserved both the HER2 antigen specificity and the natural killer cell-associated mechanism of action of trastuzumab. When administered as a tumor treatment, AAV9.trastuzumab increased median survival. Dose-escalation studies revealed that higher doses of AAV9.trastuzumab resulted in smaller tumor volumes. Our results indicate that intrathecal AAV9.trastuzumab may provide significant antitumor activity in patients with HER2-positive breast cancer brain metastases.Significance: Intrathecal delivery of trastuzumab via adeno-associated virus has the potential to become a novel, integral part of adjuvant therapy for patients with HER2-positive breast cancer brain metastases. Cancer Res; 78(21); 6171-82. ©2018 AACR.

Preparation of Nonhuman Primate Eyes for Histological Evaluation After Retinal Gene Transfer.

To evaluate gene therapy for retinal disorders, appropriate models of the human eye are needed. Nonhuman primate eyes offer significant advantages over rodent eyes. However, current preparation methods have limitations. Here, a protocol is described for histological processing of nonhuman primate eyes after gene transfer. The user dissects unfixed eyes, flattens the globe parts within filter paper, and performs formalin fixation and paraffin embedding. This method obviates the need for harsh fixatives, allowing subsequent immunostaining or in situ hybridization while preserving tissue integrity for histopathological evaluation. Moreover, the straight orientation of the retinal cell layers is ideal for image analysis.

Nonclinical Pharmacology/Toxicology Study of AAV8.TBG.mLDLR and AAV8.TBG.hLDLR in a Mouse Model of Homozygous Familial Hypercholesterolemia.

The homozygous form of familial hypercholesterolemia (HoFH) is an excellent model for developing in vivo gene therapy in humans. The success of orthotropic liver transplantation in correcting the metabolic abnormalities in HoFH suggests that the correction of low-density lipoprotein receptor (LDLR) expression in hepatocytes via gene therapy should be sufficient for therapeutic efficacy. Vectors based on adeno-associated virus serotype 8 (AAV8) have been previously developed for liver-targeted gene therapy of a number of genetic diseases, including HoFH. In preparation for initiating a Phase 1 clinical trial of AAV8-mediated LDLR gene therapy for HoFH, a combined pharmacology/toxicology study was conducted in a mouse model of HoFH. No dose-limiting toxicities were found at or below 6.0 × 1013 GC/kg. Therefore, the maximally tolerated dose is greater than the highest dose that was tested. Mild and transient liver pathology was noted at the highest dose. Therefore, the no effect dose was greater than or equal to the middle dose of 7.5 × 1012 GC/kg. The minimally effective dose was determined to be ≤7.5 × 1011 GC/kg, based on stable reductions in cholesterol that were considered to be clinically significant. This translates to a therapeutic window of ≥80-fold for the treatment of HoFH.

Non-Clinical Study Examining AAV8.TBG.hLDLR Vector-Associated Toxicity in Chow-Fed Wild-Type and LDLR+/- Rhesus Macaques.

Vectors based on adeno-associated virus serotype 8 (AAV8) have been evaluated in several clinical trials of gene therapy for hemophilia B with encouraging results. In preparation for a Phase 1 clinical trial of AAV8 gene therapy for the treatment of homozygous familial hypercholesterolemia (HoFH), the safety of the clinical candidate vector, AAV8.TBG.hLDLR, was evaluated in wild-type rhesus macaques and macaques heterozygous for a nonsense mutation in the low-density lipoprotein receptor (LDLR) gene (LDLR+/-). Intravenous infusion of 1.25 × 1013 GC/kg of AAV8.TBG.hLDLR expressing the human version of LDLR was well tolerated and associated with only mild histopathology that was restricted to the liver and sporadic, low-level, and transient elevations in transaminases. Some animals developed T cells to both capsid and the hLDLR transgene, although these adaptive immune responses were most evident at the early time points from peripheral blood and in mononuclear cells derived from the liver. This toxicology study supports the safety of AAV8.TBG.hLDLR for evaluation in HoFH patients, and provides some context for evaluating previously conducted clinical trials of AAV8 in patients with hemophilia.

Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep.

A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens.

Adeno-Associated Virus Serotype 9-Expressed ZMapp in Mice Confers Protection Against Systemic and Airway-Acquired Ebola Virus Infection.

Adeno-associated viral vectors can be used as a platform for delivering biological countermeasures against pandemic and biological threats. We show that vector delivery of two antibody components of the ZMapp product is effective in mice against systemic and airway challenge with a mouse-adapted strain of Ebola virus. This platform provides a generic manufacturing solution and overcomes some of the delivery challenges associated with repeated administration of the protective protein.

Vector serotype screening for use in ovine perinatal lung gene therapy.

PURPOSE: Successful in utero or perinatal gene therapy for congenital lung diseases, such as cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral (AAV) vector serotypes following intratracheal delivery. METHODS: Six different AAV vector serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe of perinatal sheep via bronchoscopy. At 1 week, samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real-time polymerase chain reaction (qPCR). RESULTS: Fluorescent stereomicroscopy demonstrated GFP expression in the right upper lobe following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small- and medium-sized airways following intratracheal injection of AAV1, 6, 8, 9, and rh10. However, only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40 cycles, AAV1 did not show GFP gene amplification. CONCLUSION: Adeno-associated viral vector serotypes 6, 8, 9, and rh10 demonstrated efficient GFP transgene expression at early time points, and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR.

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial.

BACKGROUND: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. METHODS: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0.9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. FINDINGS: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3.7%, 95% CI 0.1-7.3; p=0.046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. INTERPRETATION: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials. FUNDING: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.