Alpha-1 antitrypsin (AAT) deficiency is a hereditary disorder associated with mutations in the
Gene therapies to treat both aspects of the disease are currently at various stages of development. For the liver disease approaches that can be considered include ribozymes, antisense, peptide nucleic acids and small-interfering RNAs; all designed to inhibit expression of the mutant gene (recently reviewed in McLean et al., 2009). For the lung disease gene therapies using non-viral, lentiviral and adeno-associated viral approaches to express the normal gene either locally or intramuscularly have been reported (Chulay et al., 2011; Brantly et al., 2006; Flotte et al., 2007; Argyros et al., 2008; Brantly et al., 2009; Liqun Wang et al., 2009); all aim to increase AAT levels in the circulation above the deficiency threshold of 11 μM. New approaches are focused on coupling haematopoietic stem cell therapy with AAT-lentiviral gene therapy (Ghaedi et al., 2010; Argyros et al., 2008). This chapter will review the history and current state-of-the-art in these areas.
2. Gene therapies targeting ZAAT-related liver disease
There are currently no available treatments for AAT deficiency-related liver disease other than transplantation. The 5-year survival is 83% for adults and 90% for children post transplant (Kemmer et al., 2008). As an alternative to transplantation gene therapy approaches aimed at inhibiting ZAAT expression in the liver can potentially be used to stop the production of the mutant Z protein, hence prohibiting the accumulation of the protein in the liver and providing protection against liver disease. Such approaches include the use of ribozymes, small interfering RNA (siRNA) and small DNA fragments (SDF), and a number of these genetic approaches designed to downregulate ZAAT expression have been tested in animals as therapies for liver disease in AAT deficiency. To date there have been no reports on the use of zinc-finger nucleases (ZFN) or peptide nucleic acids (PNA) to treat ZAAT-related liver disease (Figure 1). ZFNs are artificial restriction enzymes that can be engineered to target specific DNA sequences and exploit a cell’s DNA repair machinery to precisely alter the genome; PNAs are synthetic DNA analogues that hybridize to complementary DNA or RNA to facilitate anti-gene or antisense inhibition, respectively (Jensen et al., 1997; Pellestor & Paulasova, 2004). It has been suggested that if ZAAT levels can be decreased to those similar or lower than MZ AAT heterozygotes there may be clinical benefit (Cruz et al., 2007) as heterozygous individuals rarely develop severe liver disease, particularly in childhood.
Ribozymes are catalytic RNA molecules capable of cleaving RNA with high specificity. Their catalytic properties were discovered almost 30 years ago (Kruger et al., 1982) and they are now known to contain both a catalytic RNA domain that cleaves a target mRNA and a substrate-binding domain that contains an antisense sequence to the target mRNA sequence that enables them to bind to their target mRNA sequence through Watson–Crick base pairing (Trang et al., 2004). The best characterised ribozymes have hammerhead or hairpin shaped active centres; both are promising gene-targeting reagents.
Hammerhead ribozymes capable of cleaving AAT mRNA have been constructed and were shown to be effective at inhibiting ZAAT expression in a human hepatoma cell line (Zern et al., 1999). In this study the hepatoma cells were also stably transduced with a modified AAT cDNA capable of producing wildtype AAT protein, but resistant to cleavage by the ZAAT-targetted ribozyme. Later a bi-functional vector was constructed, which contained both the ribozyme and the ribozyme-resistant AAT gene. Once transduced into hepatoma cells, the cells showed effective expression of the transduced AAT under conditions where the endogenous AAT gene was inhibited (Ozaki et al., 1999). Effective gene therapy for ZAAT deficiency requires stable transduction of resting hepatocytes, ideally to deliver both wild-type AAT and to inhibit production of ZAAT. Duan
Together these data show promise for the use of ribozymes for ZAAT-related liver disease. Importantly ribozymes bind to their targets with high specificity however there can be problems with their use to knockdown gene expression
2.2. Small interfering RNA
RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing. It is initiated by short double-stranded RNA (dsRNA) sequences called small
interfering RNA (siRNA) that are generated from longer transcripts by the enzyme Dicer. Each siRNA has specificity for a target RNA via its homology to sequences within the target gene (Elbashir et al., 2001). siRNAs form part of the RNA-induced silencing complex (RISC), a multi-component nuclease containing RNAse III, that enables the destruction of target mRNAs (Hammond et al., 2000). The argonaute protein within RISC incorporates one strand of an siRNA, known as the guide strand, and uses this as a template for recognizing complementary mRNA. Once found the target mRNA is cleaved by activation of the RNAse activity within RISC.
2.3. Small DNA fragments
The use of small DNA fragments (SDF) for homologous replacement of mutant genes allows targets to be directly altered by insertion, deletion or replacement. Theoretically direct conversion of the mutant sequence to a wild-type genotype ensues, thereby restoring the normal phenotype. The basis of this technology involves the introduction of small DNA fragments into cells that recombine with the genomic DNA at a targeted site, thereby producing a specific change in the sequence. The feasibility of using SDF targeting the AAT gene has been reported. SDFs encoding the normal AAT sequence were generated and transfected
3. Gene therapies to target ZAAT-related lung disease
There have been a number of gene therapy trials for the treatment of AAT deficiency-related lung disease. The first used cationic liposomes encapsulating a plasmid carrying the human AAT cDNA (Brigham et al., 2000). In this open-label study the liposomes were administered intranasally to 5 ZAAT deficient individuals. The results showed that AAT levels were increased at day 5 following administration and returned to baseline by day 14. Subsequent studies have improved on this by using non-pathogenic human parvovirus recombinant AAV vectors that are more efficient at transgene delivery and are more capable of extended transgene expression.
3.1. Intramuscular adeno-associated viral gene delivery
In 2006 Brantly
Later in another phase 1, open-label, dose-escalation clinical trial sustained AAT expression was achieved using a rAAV serotype 1 vector (Brantly et al., 2009). rAAV1 is substantially more efficient than rAAV2 in transducing skeletal muscle (Cruz et al., 2007). Once again subjects were dosed via intramuscular (i.m.) injection and those subjects who had been receiving protein therapy discontinued its use for 28 or 56 days prior to vector administration. In those who received 2.2 x 1013 and 6.0 x 1013 vector genome particles normal AAT was expressed above background in all subjects. AAT expression was sustained at levels 0.1% of normal for at least 1 year in the highest dosage level cohort. Vector administration was well tolerated and there were no changes in hematology or clinical chemistry parameters however neutralizing antibody and IFN-gamma enzyme-linked immunospot responses to rAAV1 capsid were evident in all subjects at day 14.
The most recent report on this gene therapy approach describes the results of a preclinical evaluation of a rAAV1 vector expressing human AAT made using a recombinant herpes simplex virus production method that can achieve much higher yields enabling a substantial increase in dosage in clinical studies (Chulay et al., 2011). The toxicology study in mice treated i.m. with this vector showed that the HSV-produced vector had favorable characteristics in terms of purity, efficiency of transduction, and human AAT expression.
Administration with this vector led to no significant differences in clinical findings or hematology and no gross changes in pathology although there were mild changes in skeletal muscle at the injection site. These consisted of focal chronic interstitial inflammation and muscle degeneration, regeneration and vacuolization in vector-injected animals. Vectors were detectable in blood 24 hours after dosing and declined thereafter, with no copies detectable 90 days after dosing. Antibodies to human AAT were detected in almost all treated animals, with antibodies to HSV detectable in most animals that received the highest vector dose. With higher doses of HSV-produced vector, the increase in serum human AAT levels was dose-dependent in females and, interestingly, greater than dose-proportional in males. Together all of these studies support continued development of rAAV1-AAT vectors for i.m. gene delivery for the treatment of AAT deficiency (Figure 2).
3.2. Viral gene delivery to the lung
Transduction of cells within the lung is an attractive approach for AAT gene therapy. Since somatic tissues are comprised of heterogeneous, differentiated cell lineages that can be difficult to specifically transfect (although this may have been largely overcome by the work of Flotte using i.m. injection (section 3.1)), methods have been tested to deliver gene therapies directly to cells within the lung. These include AAV and other viral methods to deliver the AAT gene directly to cells within the lung for local expression of AAT at the site where it is most needed to provide an antiprotease protective screen. Both integrating lentiviral systems and non-integrating AAV vectors are capable of gene transfer and expression
3.3. Stem cell therapy and other approaches to gene delivery
An ideal gene therapy approach should enable persistent transgene expression without limitations of safety and reproducibility. Using an individual’s own cells, cultured
Human induced pluripotent stem cells (iPSC) also hold great promise for cell-based therapy and in cell studies of genetic diseases. A strategy using dermal fibroblasts from AAT-deficient individuals has generated a library of patient-specific human iPSC cell lines (Rashid et al., 2010). Once differentiated into hepatocytes the resulting cells exhibit properties of mature hepatocytes and recapitulate key pathological features of AAT deficiency including accumulation of misfolded AAT in the endoplasmic reticulum. Using an approach such as this it may be possible to generate iPSC hepatocytes from an AAT-deficient individual, repair the defect
The next step in advancing this technology will be to achieve this without the worry of endogenous effects mediated by reprogramming transgenes. A number of methods already exist for generating murine or neonatal iPSCs free of reprogramming transgenes however, until recently this was not the case for disease-specific iPSC from humans with inherited or degenerative diseases. Now a humanized version of a single lentiviral "stem cell cassette" vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans has been developed (Somers et al., 2010). The human iPSCs generated using this vector contain a single viral integration that can excise to generate human iPSCs free of integrated transgenes. As a proof of principle, this strategy has been used to generate lung disease-specific iPSC lines from individuals with AAT deficiency-related emphysema. These cells have the ability to differentiate into developmental precursor tissue of lung epithelia and will prove invaluable for AAT-deficiency related investigations. However the most exciting aspect of this technology is its potential to generate safer autologous ‘corrected’ cell therapies similar to those described above.
It is sometimes suggested that non-viral episomal plasmid DNA (pDNA) vectors may offer some advantages over viral vectors in that they can be produced cheaply and in large quantities. Argyros
Since the first human gene therapy trials using lymphocytes for the cellular delivery of the adenosine deaminase gene to patients with severe combined immune deficiency (SCID) and as tumour-infiltrating vehicles there has been a remarkable expansion in the development of gene therapy strategies (Culver et al., 1991a; 1991b; 1991c). The most promising data have come from therapies targeting monogenic disorders. With respect to ZAAT deficiency, rAAV2 vectors, and more recently rAAV1 due to its higher tropism for muscle cells, have shown great promise for delivery of the AAT gene. The advantages of AAV vectors include not only their lack of acute pathology but also their episomal nature. Occasional problems with adaptive immune responses to its capsid proteins have been encountered in some contexts (Manno et al., 2006), however newer generation vectors with greater safety and efficiency of gene transfer are constantly under development. Non-AAV gene therapy strategies for AAT-related lung disease, although less advanced, also show potential. Genetic therapies designed to down-regulate expression of ZAAT in hepatocytes to reverse the toxic gain of function in the liver are also less well developed, however the next decade is likely to see huge progress in this area. Finally the recent advent of various stem cell technologies can only enhance efforts in ZAAT deficiency and gene therapy research.
Funding for alpha-1 antitrypsin deficiency research in this department is gratefully acknowledged from the US. Alpha One Foundation, the Medical Research Charities Group and the Health Research Board of Ireland, Programmes for Research in Third Level Institutes administered by the Higher Education Authority, the Children’s Medical and Research Centre, Crumlin Hospital and the Department of Health and Children.