Dragana Vidovic

rAAV-CFTRΔR Rescues the Cystic Fibrosis Phenotype in Human Intestinal Organoids and Cystic Fibrosis Mice

RATIONALE Gene therapy holds promise for a curative mutation-independent treatment applicable to all patients with cystic fibrosis (CF). The various viral vector-based clinical trials conducted in the past have demonstrated safety and tolerance of different vectors, but none have led to a clear and persistent clinical benefit. Recent clinical breakthroughs in recombinant adeno-associated viral vector (rAAV)-based gene therapy encouraged us to reexplore an rAAV approach for CF. OBJECTIVES We evaluated the preclinical potential of rAAV gene therapy for CF to restore chloride and fluid secretion in two complementary models: intestinal organoids derived from subjects with CF and a CF mouse model, an important milestone toward the development of a clinical rAAV candidate for CF gene therapy. METHODS We engineered an rAAV vector containing a truncated CF transmembrane conductance regulator (CFTRΔR) combined with a short promoter (CMV173) to ensure optimal gene expression. A rescue in chloride and fluid secretion after rAAV-CFTRΔR treatment was assessed by forskolin-induced swelling in CF transmembrane conductance regulator (CFTR)-deficient organoids and by nasal potential differences in ΔF508 mice. MEASUREMENTS AND MAIN RESULTS rAAV-CFTRΔR transduction of human CFTR-deficient organoids resulted in forskolin-induced swelling, indicating a restoration of CFTR function. Nasal potential differences demonstrated a clear response to low chloride and forskolin perfusion in most rAAV-CFTRΔR-treated CF mice. CONCLUSIONS Our study provides robust evidence that rAAV-mediated gene transfer of a truncated CFTR functionally rescues the CF phenotype across the nasal mucosa of CF mice and in patient-derived organoids. These results underscore the clinical potential of rAAV-CFTRΔR in offering a cure for all patients with CF in the future.

Immunological Ignorance Allows Long-Term Gene Expression After Perinatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer to Murine Airways

Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and α(1)-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months.

A novel translational model for fetoscopic intratracheal delivery of nanoparticles in piglets

The aim of this study was to assess the feasibility of fetal tracheal injection in the late‐gestational pig to target the airways.

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome(1,2) or hyperoxic injuries of the neonatal lung(3). Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)(4), genetic variants of surfactant deficiencies(5) and α1-antitrypsin deficiency(6). Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individuals immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies(7). In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep(8), and even in a clinical setting(9), but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size. Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection(10-12), (ultrasound-guided) intrapulmonary injection(13,14) and intravenous administration into the yolk sac vessels(15,16) or umbilical vein(17). Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques(18).

Roadmap for an early gene therapy for cystic fibrosis airway disease

Gene therapy provides a mutation‐independent approach to treat or even cure CF airway disease. To develop a clinical candidate for CF gene therapy, a thorough examination of preclinical efficacy in relevant cell and animal models is a prerequisite. For a long time, the CF field was struggling with a lack of appropriate animal models for CF airway pathology. Since 2008, many different and complementary animal models have been generated that develop hallmarks of CF airway disease, including the CF pig, ferret, and rat. With this, a new era has arisen that allows investigating the efficacy of gene therapy beyond molecular and electrophysiological end‐points. Successful gene therapy most likely requires an appropriate time window. CF lung pathology progresses with age and therefore an early treatment would be beneficial to prevent irreversible damage. In that regard, newborn screening programs and prenatal diagnosis already provide a basis to facilitate future preventive gene‐based treatment. If successful, gene therapy for CF airway disease would markedly reduce the treatment burden and improve life quality and life expectancy of CF patients.

The phospholipid flippase ATP8B1 mediates apical localization of the cystic fibrosis transmembrane regulator

Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.

WS01.1 rAAV2/5 encoding a truncated CFTR rescues the CF phenotype in intestinal organoids and a CF mouse model

To date, the majority of CF patients receive symptomatic treatment. Gene therapy offers the potential to cure CF in mutation-independent manner. Here, we evaluate rAAV2/5 gene therapy for CF in human intestinal organoids and a ΔF508 mouse model. First, we studied the longevity of gene expression after a single administration of a rAAV2/5-Fluc to mouse airways. This resulted in relatively stable gene expression up to 15 months with only a 4-fold decrease in bioluminescent signal in lungs. Due to the rAAV size limit, we used a truncated CFTR missing a portion of the R-domain, CFTRΔR (Ostedgaard et al., PNAS, 2005). First, we evaluated its functionality in a physiological relevant and highly translational organoid model. These data demonstrate that the therapeutic vector rAAV-CFTRΔR is functional and that the ion transport-induced organoid swelling is CFTR-specific. Indeed, despite the expression cassette being at the limit of the rAAV packaging capacity, intact genomes are incorporated into vector particles, that allow efficient second strand DNA synthesis and transgene expression. Finally, we administered rAAV2/5-CFTRΔR to ΔF508 mice by nasal instillation. 2–4 weeks later, we demonstrated a response to low-chloride and forskolin perfusion in 6 out of 8 mice by nasal potential differences, indicating restoration of chloride transport across nasal mucosa. In conclusion, we obtained sustained reporter gene expression in murine airways and demonstrated restoration of the CF phenotype in CF organoids and a CF mouse model. Our results underscore the therapeutic potential of rAAV2/5-CFTRΔR for CF gene therapy opening new avenues towards a definitive cure for all CF patients.