Gene Therapy for Cystic Fibrosis Paved the Way for the Use of Adeno-Associated Virus in Gene Therapy

Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). ¹ In CF patients, failure of CFTR to produce a thin layer of fluid causes sticky mucus secretions, leading to chronic lung infection and inflammation, gastrointestinal (GI) obstruction, male infertility, liver disease, and failure to digest food as a result of the loss of pancreatic duct function. ^2,3 Since CF is a single gene, carriers bearing only one CF mutation are normal, making CF a prime candidate for the development of a gene therapy. ^4,5 Indeed, repair by gene therapy was demonstrated early on by a restoration of chloride channel function in vitro. ⁶ Even though many organs are affected, defective airways are the most frequent cause of mortality; thus, developing a gene therapy based on repairing airway disease became an early priority. ^7,8

Early Days of Gene Therapy

In the minds of some of those who were pioneers in the field, the overall concept of gene therapy seemed simple: replace a dysfunctional gene and restore function. ⁹ Given that viruses have evolved to infect cells and transfer genetic components, it was logical to employ a modified virus as a transfer agent to introduce a replacement gene into the cells of a malfunctioning organ. ¹⁰ The lung is ideal for this purpose because respiratory virus infection is common, and modified viruses could potentially be used to deliver genes to airway cells. ¹¹ To others, the prospect of using a virus as a gene transfer agent raised many concerns, including ethical issues associated with altering the human genome. Concerns and issues related to recombinant DNA technology were deemed so important that an NIH Recombinant DNA Advisory Committee (RAC) was charged with reviewing them. ¹² Early work to develop a gene therapy for CF provided much of the groundwork that was reviewed by the RAC and alleviated many of the early concerns regarding gene therapy. ¹³ Although many of the original concerns have been resolved, some are still being discussed. ¹⁴

Adeno-Associated Virus

Among the early viral vectors chosen for gene therapy was adeno-associated virus (AAV), ¹¹ a defective parvovirus isolated from humans and primates. ¹⁰ Following infection, this wild-type DNA virus undergoes site-specific stable integration into human chromosome 19. ¹⁵ It remains dormant even after being integrated into the genome, requiring co-infection with a helper adenovirus or herpesvirus for replication. ¹⁶ The AAV genome is composed of a 4.68-kb, linear single strand of DNA consisting of inverted terminal repeats (ITRs) ¹⁷ and rep and cap proteins. The ITRs represent 145 nt of DNA and are absolutely required for integration, replication (ori), excision, and packaging. The rep gene is involved in replication; there are four rep proteins produced by alternate splicing. ¹⁸ The cap gene is required for encapsidation; in this case, there are three proteins, also produced by alternate splicing. ¹⁹ Single-stranded AAV must be converted into a double-stranded form for gene expression. ²⁰

Recombinant AAV Production

AAV is advantageous for gene therapy in that it has only two sets of genes, involved in replication and capsid formation. ^19,21 Both these sets of genes are removed to produce a recombinant virus. To produce recombinant AAV for CF gene therapy, three components are needed: (1) the CFTR cDNA, subcloned into a plasmid flanked on both the 5′ and 3′ ends with ITRs; (2) a second plasmid containing the rep and cap genes; and (3) a method for providing the helper function associated with the adenovirus. ²² In the earlier days of AAV gene therapy, AAV was produced by triple transfection, that is, transfecting with all the three of these components in separate plasmids; more recently, scalable production methods have become much more sophisticated, reducing the number of plasmids necessary to produce large quantities of AAV (see the study of Flotte et al. ²² as an example). An early version of AAV used in gene therapy contained the ITR-CFTR-ITR coding sequence encapsulated within the viral capsid proteins. It was an engineered vector based on AAV serotype 2 and referred to as tgAAV2-CFTR ²³ (tg = targeted genetics). It contained the full-length coding sequence of CFTR subcloned between the AAV2 ITRs. The tgAAV2-CFTR vector contains a synthetic polyadenylation signal based on the murine β-globin gene. This vector utilizes the intrinsic promoter of the AAV2 ITR ⁷ to drive CFTR expression, making it possible to package the full-length CFTR (4,400 nt). The idea of using the intrinsic AAV promoter was based studies of the mRNA expression of CFTR in normal airways, which is ≈1–2 copies per cell. ²⁴ Thus, inclusion of a weak promotor from AAV to drive low level expression of CFTR was thought to be physiologic. In hindsight, the weak promoter in the tg-vector was considered one of the causes of lack of clinical efficacy and new more powerful promoters were developed. ²⁵

Given that single-stranded AAV must be converted into a double-stranded form for gene expression, ²⁰ soon after recombinant CFTR containing AAV vectors were constructed, the issue was raised whether they would enter epithelial cells and whether the single- to double-strand conversion could occur and rescue defective CFTR function. This question was addressed initially in vitro, where it was demonstrated that expression of recombinant CF genes using AAV vectors in CF bronchial epithelial cells corrects defective Cl⁻ secretion and induces the appearance of small linear conductance Cl⁻ channels characteristic of CFTR. ⁶ Subsequent studies in animals showed that entry into epithelia cells in vivo using the tgAAV2-CFTR was inefficient and that other serotypes including AAV5 and AAV1. ^26,27

Although the tgAAV2-CFTR ²³ did not contain the rep and cap genes, a considerable worry regarding AAV gene therapy was whether the recombinant AAV vectors would integrate into genome. However, studies that examined the fate of rep-deleted vector DNA indicated that the ITR-CFTR-ITR coding sequence is located episomally or integrated randomly at a low frequency in the host cells, ²⁸ leading to long-term expression of the recombinant protein in the infected cells. The low frequency of integration of the recombinant vector remained a worry, but in the animal and human studies outlined below did not cause overt pathology.

Understanding the Safety Profile of AAV2-CFTR Vectors

A number of preclinical studies were performed to define the safety of recombinant AAV2-based vectors. In one study, primary cultures of cells isolated from nasal polyps of CF patients were infected with rAAV2-CFTR. The results demonstrated efficient transduction of CFTR, as assayed by detecting recombinant viral CFTR-containing DNA using vector-specific polymerase chain reaction and immunofluorescent staining for CFTR. ⁸ In another early study, rAAV2-CFTR was instilled into the bronchi of rabbits via a bronchoscope. Amazingly, CFTR RNA and protein were detected in the bronchi for up to 6 months. In a subsequent study, Rhesus macaques were treated with a single dose of rAAV2-CFTR by bronchial administration using a protocol similar to that described earlier for rabbits. ²⁹ Remarkably, vector-specific DNA and rRNA expression was detected for up to 180 days after infection. These early animal studies showed that long-term expression of CFTR following a single dose of vector is feasible. Most importantly, there were no indications of inflammation or other toxicity.

Can AAV-CFTR Vectors be Readministered?

Given that airway cells are normally exposed to the environment and are subjected to infection, they turn over and are replaced by new cells derived from basal cells. ^30,31 Thus, a gene therapy designed to treat airway cells would have to be readministered periodically. The key hurdle for repeat dosing has been the development of neutralizing antibodies (reviewed in Refs. ^32,33 ). The hypothesis that repeated dosing is feasible was tested in New Zealand white rabbits and Rhesus monkeys, ^34,35 using two doses of AAV2-CFTR followed by a single dose of either rAAV2-CFTR or green fluorescent protein (GFP). In the rabbits, the presence of neutralizing antibodies in serum increased after each dose. However, despite this increase in antibodies, GFP expression was detected 3 weeks after the end of the experiment. A similar protocol was used for the Rhesus macaques, which received ∼10¹³ DNase-resistant particles (DRP) per dose. At the end of the experiment, GFP expression was again detected, despite the presence of escalating titers of neutralizing antibodies. Somewhat disappointing was the finding that repeated dosing caused a significant drop in the ability to detect vector-derived mRNA, possibly indicating that repeated dosing reduces the magnitude of vector transduction. A common conclusion from both the rabbit and the monkey studies was that repeated dosing with AAV is safe.

The First Human Trials of AAV Vectors

In the first human AAV clinical trial for the treatment of CF, tgAAV2-CFTR was instilled into one of the maxillary sinuses. ³⁶ This mode of instillation was considered the safest way to test a virus in humans. In this trial, the recombinant AAV2 virus was administered to 10 pancreas-insufficient patients with CF. The highest levels of gene transfer were observed 2 weeks after treatment: levels in the range of 0.1 to 1 AAV-CFTR vector copy per cell. The vector persisted for as long as 10 weeks after treatment. To assess the correction of the defective CFTR, functional assays were performed in the patients' sinuses, and restoration of function as a result of tgAAV2-CFTR treatment was detected. No toxicity was seen to result from the treatment. This study was significant because it demonstrated successful infection with tgAAV2-CFTR and its persistence and transduction in the sinuses, with no evidence of toxicity. Most important was the fact that correction of the dysfunctional CFTR was also observed. Given these promising results, a Phase II, double-blinded, randomized, placebo-controlled clinical trial was conducted in 23 CF patients. ³⁷ In this trial, the primary endpoint was the rate of relapse of clinically defined sinusitis within a 3-month follow-up. Unfortunately, the rate of sinusitis did not differ significantly between the placebo and treated groups, and no improvement in CFTR function was detected.

With the barriers regarding the safety of using recombinant AAV vectors in humans surmounted, a first trial was initiated with rAAV in human lungs: a Phase I study in 25 adult and adolescent CF patients with mild-to-moderate lung disease, in which the dose of tgAAV2-CFTR ranged from 3 × 10¹ to 1 × 10⁹ replication units (RU), equivalent to ∼6 × 10⁴ to 2 × 10¹² DRP. ³⁸ This first trial was followed by a Phase I, single-administration, dose-escalation trial in which the tgAAV2-CFTR was administered by nebulization to the lungs of CF subjects. ²³ The procedure in this trial differed from the single-dose trials in which the vector was applied directly or via bronchoscopic delivery. As was true for the previous studies, administration was deemed to be safe (the primary outcome in this Phase I study).

Given the positive safety data from the single-dose studies, two repeat-dosing clinical trials were performed using the tgAAV2-CFTR vector in CF patients. The first was a randomized, double-blind, placebo-controlled Phase II trial. ³⁹ This trial was followed by a Phase IIB study in which 102 subjects older than 12 years were treated with two doses of 1 × 10¹³ DRP of tgAAV2-CFTR or corresponding placebo, administered 30 days apart. ⁴⁰ Although safety was once again documented, the study did not meet its primary endpoint of statistically significant improvement in lung function over placebo at 30 days after the initial administration of tgAAV2-CFTR.

A Successful Beginning

Although these studies did not produce a treatment for CF, they still represent pioneering work in many ways. For example, the original animal studies described above were conducted using recombinant virus engineered and produced in university laboratories. These were small-scale studies using laboratory-grade virus. Vector production facilities at the Targeted Genetics Corporation, with Barrie Carter as its Chief Scientific Officer, had to be designed and built to produce large quantities of virus. For example, hundreds of doses of 1 × 10¹³ DRP of virus suitable for use in humans were produced, requiring a significant scale-up in virus production. During that time, the parameters of what was considered a good manufacturing practice-level virus suitable for use in humans also had to be defined in partnership with the Food and Drug Administration. Finally, unlike the animal studies in which short-term safety was already known, both the short- and long-term risks of applying AAV virus to humans and the possible contamination of the associated health care workers had to be evaluated in these studies.

Thus, although the many clinical studies of AAV have been widely interpreted as disappointing, the data can also be seen as groundbreaking, indicating that with improved vector transfer and improved gene expression, gene therapy for CF is nevertheless feasible.