Cell-specific delivery of genes is an attractive and desirable strategy for inherited rare diseases for its potential curative outcomes. Thus, adeno-associated virus (AAV) vectors have been increasingly used as delivery vehicles for developing gene therapies. Although AAVs have limited cargo capacity (i.e., < 5kb), their use in gene therapy reduces two significant concerns associated with other viral vectors, such as genomic integration and adverse immune reactions. Additionally, their specific tropism and transduction properties have already enabled successful treatment of various inherited conditions, including retinal diseases (e.g., Luxturna-AAV2), spinal muscular atrophy (e.g., Zolgensma-AAV9), and lipoprotein lipase deficiency (e.g., Glybera-AAV1, removed from the market due to limited use) (Domenger and Grimm, 2019).
Several gene therapies for rare CNS diseases (e.g., Giant Axonal Neuropathy-GAN, Angelman Syndrome, and Aromatic L-Amino Acid Decarboxylase Deficiency-AADC) are currently at different stages of preclinical and clinical evaluation that leverage AAV’s unique tropism to target neurons (Mendell et al. 2020). Among these, a recent report on the clinical success of an AAV2-based gene therapy for ADCC illustrates the continued growth and evolution of this approach.
With only over 100 cases documented globally, AADC deficiency is a very rare autosomal recessive neurometabolic syndrome. Defects in AADC activity occur due to mutations in the DCC gene, which lead to deficits in the synthesis of the neurotransmitters serotonin and dopamine. Additionally, as dopamine is a precursor for synthesizing norepinephrine and epinephrine, AADC patients are also deficient in these neurotransmitters.
Main physiological reactions of Dopa decarboxylase (DDC). Decarboxylation of L-Dopa and L-5-HTP catalyzed by DDC. Retrieved from Montioli and Voltattorni, 2021 without modifications.
Individuals carrying DCC disease-causing mutations are afflicted from infancy. Phenotypes may vary from mild to very severe deficiencies in motor function and decreased muscle tone. Oculogyric crises, or involuntary eye movements typically upwards, are common in AADC patients (Solberg and Koht, 2017). Other common phenotypes include mood and sleep disorders.
Cystic fibrosis is an autosomal recessive disease that results from mutations in the CFTR gene (cystic fibrosis transmembrane conductance regulator). Over 2,000 mutations have been identified which affect CFTR’s expression or function through different mechanisms. Disease severity and time of onset depend on the specific underlying mutations and the extent of CFTR loss of function. A hallmark of cystic fibrosis is the increased sweat salt concentration, which serves as a diagnostic criterion (i.e., sweat chloride analysis). Phenotypes associated with severe disease and directly responsible for cystic fibrosis morbidity include increased accumulation of thick mucus secretions in airways, chronic inflammation, and susceptibility to recurrent respiratory infections (De Boeck, 2020).
CFTR is an ATP-gated anion channel critical for transepithelial fluid and electrolyte transport in respiratory, gastrointestinal, and pancreas tissues, among others (Hwang et al. 2018). CFTR localizes to the apical epithelium of exocrine glands, where it helps maintain ion concentration balance. Besides its chloride conductance, CFTR modulates the epithelial sodium channel activity (ENaC) (Berdiev et al. 2009). As it occurs due to mutations associated with cystic fibrosis, the malfunction of CFTR results in thickening of glandular secretions and consequent duct obstruction and atrophy (e.g., airways).
Reprinted from “Cystic Fibrosis Airways”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates
The most prevalent CFTR mutation, F508del, leads mostly to complete loss of channel function due to protein misfolding and defective channel gating (Patel et al. 2019). There is no cure for cystic fibrosis, and available therapies traditionally have only ameliorated disease symptoms. More recently, small molecules have been used that target CFTR and enhance its function. Nevertheless, the effectiveness of these modulators is highly dependent on the specific CFTR mutation(s) and other genetic and epigenetic factors, which may impact individual responses. Therefore, these therapies do not provide relief to all patients.
Recognizing the need for curative therapies to restore CFTR function effectively, the Porteus lab has initiated studies combining the use of CRISPR/Cas9 and AAV vectors to achieve gene editing of airway basal stem cells. In previous work, their efforts focused on repairing the most prevalent mutation associated with cystic fibrosis, the F508del (Vaidyanathan et al. 2020). Nevertheless, because of the heterogeneity of mutations underscoring cystic fibrosis, Porteus and colleagues have envisioned a strategy for targeted replacement of the full CFTR sequence at its endogenous locus. Once edited ex vivo, airway basal stem cells derived from cystic fibrosis patients may be used for autologous cell therapy. By using two different AAV vectors, each carrying part of the CFTR sequence, Vaidyanathan and colleagues at the Porteus lab have been able to bypass the payload size limitations of AAVs (Vaidyanathan et al. 2021). This approach also allowed the team to include sequences corresponding to homology arms to enable homologous recombination events and a specific tag sequence to enrich gene-edited airway basal stem cells.
Following this strategy, Vaidyanathan et al. successfully inserted the full CFTR sequence, modifying airway basal stem and human bronchial epithelial cells derived from eleven cystic fibrosis patients. Correction of CFTR expression was confirmed by immunoblotting, and functional recovery was demonstrated by improved transepithelial ion transport in differentiated epithelial sheets derived from edited cells (Li et al. 2004). Furthermore, recovery of CFTR chloride transport was as high as 150% compared to non-disease control cells.
Therefore, the Poarteus lab has developed a CRISPR/Cas9-AAV method that supports the correction of all CFTR mutations and thus may provide a solution to a broader patient population through autologous airway stem cell therapy. Nevertheless, the team acknowledges the significant challenges ahead for developing methods to implant CFTR corrected stem cells into airway epithelia, a process that they are prepared to initiate by using biomaterial vessels in animal models of cystic fibrosis sinus disease.
In a new approach to the treatment of AADC deficiency, recently, investigators leveraged an AAV2 vector to target two midbrain regions, substantia nigra pars compacta (SNc) and ventral tegmental area (VTA), for delivery of the normal human AADC sequence (hAADC). Teams from Neurological Surgery Departments at the University of California San Francisco and Ohio State University designed the clinical study (ClinicalTrial NCT02852213) to evaluate the feasibility and safety of this new gene therapy strategy.
Previous clinical studies in Japan and Taiwan had targeted the putamen for AAV2-hAADC delivery (Hwu et al. 2012, Kojima et al. 2019). However, Pearson and colleagues believed that patients could achieve more significant benefits in improved motor function and behavioral and autonomic symptoms by restoring AADC activity and specifically dopamine biosynthesis in the SNc and VTA (Pearson et al. 2021).
Distribution of dopamine in the human brain. Pearson and colleagues targeted the SNc and VTA with AAV2-hAADC to restore dopamine biosynthesis and neurotransmission in the nigrostriatal, mesolimbic, and mesocortical pathways. Adapted from “Distribution of Catecholamine Neurotransmitters in the Human Brain”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates
A total of seven pediatric patients with severe motor disabilities participated in the study and received magnetic resonance imaging-guided infusions of the AAV2-hAADC vector directly into the SNc and VTA. Pearson and colleagues confirmed successful AADC expression through increased tracer (FDOPA) uptake in targeted brain regions and dopamine metabolites in cerebrospinal fluid, which persisted after one and two years following gene therapy, respectively. Consistent with improved AADC activity, Oculogyric crises ended in most participants within a month after gene therapy. Motor functions such as head control and independent sitting were substantially improved one year after gene therapy in most participants. Significantly, two participants were able to perform supported walking a year after treatment. Lastly, AAV2-hAADC gene therapy improved behaviors such as mood, sleeping patterns, and feeding tolerance.
Overall, investigators found that AAV2-hAADC based gene therapy was safe and substantially contributed to improved quality of life for participants and their families. In addition, a significant advantage of this approach is the ability of AAV2-hAADC to transduce neurons in other brain regions through anterograde transneuronal transport, which investigators believe may have contributed to the positive outcomes of this study (Salegio et al. 2013).
