Review

Current status of gene therapy for a-1 antitrypsin deficiency Heather S Loring & Terence R Flotte† 1.

Introduction

University of Massachusetts Medical School, Gene Therapy Center, Worcester, MA, USA

2.

AAT protein replacement

3.

Vectors for AAT gene therapy

4.

Preclinical and clinical

Introduction: As a common monogenic disease, a-1 antitrypsin (AAT) deficiency has undergone thorough investigation for the development of gene therapy. The most common pathology associated with AAT deficiency occurs in the lung, where the loss of function due to impaired secretion of mutant AAT prevents the inhibition of neutrophil elastase and leads to loss of elastin content from the alveolar interstitium. Areas covered: Current treatment in the USA consists of recurrent intravenous protein replacement therapy to augment serum AAT levels. In an attempt to replace recurring treatments with a single dose of gene therapy, recombinant adenovirus, plasmid, and recombinant adeno-associated virus (rAAV) vectors have been investigated as vectors for transgene delivery. Expert opinion: Large strides in gene therapy for AAT deficiency lung disease have led to the development of rAAV1-AAT capable of producing sustained serum AAT levels in clinical trials after intramuscular administration in humans at 3% of the target level. Further increases in levels are anticipated as limb perfusion targets greater muscle mass. The future roles of intrapleural and airway delivery, miRNA-expressing vectors, iPS cell platforms, and genome editing are anticipated.

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development 5.

Conclusion

6.

Expert opinion

Keywords: adeno-associated virus, a-1 antitrypsin, emphysema, gene therapy, liver disease, vectors Expert Opin. Biol. Ther. [Early Online]

1.

Introduction

a-1 antitrypsin (AAT) deficiency is among the most common monogenic disorders affecting the lung. The missense mutant form of AAT (E342K), described as PiZ based on its electrophoretic mobility on isoelectric focusing gels, is remarkably common among Northern European populations, with allele frequencies ranging from 5 to nearly 25%. The PiZ allele frequency in North America is ~ 3%. It was first described in 1963 by Laurell and Ericsson when they observed the absence of the a-1 globulin band on serum protein electrophoresis in cohorts of patients with familial clustering of emphysema [1-4]. AAT normally functions as a circulating serum anti-protease, being a member of the serine proteinase inhibitor (serpin) family. It is a 52 kD glycoprotein, normally produced within hepatocytes, and monocytes to a lesser extent, and secreted into the plasma, where it is the second most abundant circulating serum protein. AAT normally serves to inactivate neutrophil elastase (NE) and multiple other proteases and pro-inflammatory neutrophil-derived products (such as cathepsins, proteinase-3, and a-defensins) [5-10]. Severe AAT deficiency (total plasma levels < 11 microM or 57 mg/dl) occurs in patients with two mutant alleles and results in the spontaneous development of lung disease in adulthood caused by low serum AAT concentrations [5,11,12]. In the case of the common PI*Z (E342K) mutant, the disruption of a salt bridge in the b-sheet structure allows for polymerization as the reactive loop of one molecule of Z-AAT inserts into the b-sheet of an adjacent one [13-16]. After loop-sheet polymerization, 10.1517/14712598.2015.978854 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

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H. S. Loring & T. R. Flotte

Article highlights. . . . .

a-1 antitrypsin (AAT) deficiency is a common single gene disorder. AAT protein replacement provides a proof-of-principle for gene therapy. rAAV1-AAT is one gene therapy product undergoing phase 2 clinical trials for AAT lung disease in the US. Other promising gene therapy technologies include pleural delivery and dual function vectors.

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This box summarizes key points contained in the article.

secretion from hepatocytes is rendered inefficient, leading to serum deficiency [13,17]. Common lung disease phenotypes include emphysema, chronic obstructive pulmonary disease and asthma [18-20]. Patients with severe deficiency who are either PI*ZZ homozygotes or compound heterozygotes can also develop liver disease as a result of mutant AAT polymerization within the hepatocytes [21-24]. 2.

AAT protein replacement

Around 1980, Gadek et al. recognized the imbalance of the primary neutrophil-derived protease, NE, relative to the primary circulating antiprotease, AAT, in the lower respiratory tracts of PI*ZZ homozygotes [25-27]. These investigators proposed that excess proteolytic activity of NE due to low serum AAT was the cause of progressive destruction of alveolar epithelium culminating in emphysema and could be corrected by therapeutically augmenting serum AAT concentrations and initiated testing of that hypothesis [12,25]. Weekly intravenous administration of 4 g AAT purified and concentrated from human plasma allowed AAT serum concentrations to be maintained on average at ‡ 70 mg/dl for 4 weeks. Bronchoalveolar lavages indicated that an increase in serum AAT concentrations by 18 mg/dl allowed for a 10% decrease in NE activity, thereby indicating that AAT replacement therapy sufficiently protects the alveolar epithelium and lung interstitium from future proteolytic degradation [25,28-30]. A serum threshold AAT concentration of at least 57 mg/dl adequately protects alveolar epithelium from NE activity; however, a serum concentration of < 80 mg/dl, in particular < 50 mg/dl, is associated with an increased risk of emphysema [10]. Crystal and Hubbard determined that weekly infusions of 60 mg/kg of AAT were sufficient in achieving long-term serum concentrations of 80 mg/dl [31]. Decreased levels of NE activity in the alveolar epithelium were correlated with AAT administration. Analysis of epithelial lining fluid of treated PI*Z homozygotes indicated that the serum AAT diffused from the vasculature into the alveolar epithelium supporting its validity as a therapeutic treatment. More recently, it has been shown by the McElvaney laboratory that weekly IV protein replacement decreases indices of degranulation in 2

peripheral circulating neutrophils and decreases the levels of anti-neutrophil cytoplasmic antibodies in this disease [32]. Augmentation therapy consisting of weekly intravenous infusions of 60 mg/kg AAT in PI*null individuals resulted in 80 mg/dl serum concentrations and notably did not initiate an immune response [33]. As augmentation therapy promised to prevent the future destruction of the alveoli caused by the hyperactivity of NE, Hubbard et al. sought to consolidate the weekly infusions into monthly infusions [31,34]. Hubbard found that intravenous administration of 250 mg/kg AAT was safe and resulted in high peak levels [34]. However, trough levels were below the protective threshold for several days at the end of each dosing interval. Therefore, this regimen is not currently considered standard practice. Due to the cost and efficiency drawbacks of intravenous augmentation therapy, Crystal and Hubbard evaluated aerosol therapy as an alternative delivery mechanism [35-38]. Plasma AAT was aerosolized into < 5 µm diameter droplets with the droplet size optimized for deposition in the alveoli while maintaining sufficient enzymatic activity. Administration of 100 mg of nebulized AAT twice daily over a week augmented AAT concentrations in alveolar epithelial lining fluid to normal levels. 3.

Vectors for AAT gene therapy

Patients receiving exogenous protein replacement therapies have exhibited tolerance for high levels of AAT without immunologic complications, perhaps indicating that the disease may be an attractive target for gene therapy without the need for tight control [5]. Furthermore, the expense and invasiveness of weekly IV AAT protein replacement therapy sparked investigation into other therapeutic methods. As a monogenic disorder with a short DNA coding sequence, AAT deficiency is an attractive target for gene therapy. Several gene transfer methods such as recombinant adenoviruses (rAds), plasmids, and recombinant adeno-associated viruses have been investigated for implementation in treating AAT deficiency [39]. Recombinant adenovirus Early on, rAd vectors demonstrated robust transgene delivery to the lungs and liver of animal models, attracting much attention for potential AAT gene therapy. Rosenfeld et al. demonstrated the first in vivo gene transfer with rAd in 1991 in the context of delivering human AAT (hAAT) to the respiratory epithelium of cotton rats. Expression of hAAT mRNA was detected in the respiratory epithelium at early time points, and hAAT protein was found in epithelial cells and bronchoalveolar lavage fluid [40]. Kay et al. transduced mouse hepatocytes in vivo with rAd-AAT, finding that nearly 100% of hepatocytes were transduced with multiple viral copies per cell while achieving therapeutic concentrations of AAT [41]. A major drawback of rAd gene delivery 3.1

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Current status of gene therapy for a-1 antitrypsin deficiency

mechanism is that innate immunity to the vector causes the host to mount a cytokine response reducing the safety and efficacy of rAd as a vector [39]. Plasmids In 1989, Brigham et al. first described the implementation of cationic liposomes complexed to plasmids as a novel in vivo DNA delivery mechanism both intravenously and intratracheally [42]. Plasmid-liposome complexes containing AAT gene administered to New Zealand White rabbits resulted in transfection (TFX) of both vascular endothelium and airway epithelium when administered intravenously whereas when the aerosolized transgene was introduced, evidence of AAT was only present in the vascular endothelium [43]. In 2000, Brigham et al. aerosolized AAT plasmid--liposome complexes and delivered the gene to one nostril with the other nostril acting as the control in five patients [44]. According to nasal lavage fluid (NALF) analysis, AAT expression increased in the transfected nostril to one-third of the normal level, but was unchanged in the control nostril. Levels of interleukin-8 in NALF decreased after TFX indicating that AAT gene therapy has anti-inflammatory properties. The application of AAT plasmid--liposome complexes proved to be both a safe and effective method for gene delivery to the lungs.

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3.2

Recombinant adeno-associated virus Recombinant adeno-associated viruses (rAAV) are promising for human gene therapy because of their natural inclination to persist in human cells without causing pathology [45,46]. AAVs are single-stranded DNA viruses with icosahedral capsids that necessitate an accessory virus, such as adenovirus for replication [45,47]. Without the accessory virus, AAV is dormant within the host until an accessory virus stimulates replication and site-specific integration. Since the time of first published success with rAAV2 as an in vivo vector in 1993 [48], rAAV have been adapted to deliver certain transgenes to a number of different cell and tissue types [39]. Originally, rAAV2 was widely used because of its historical role in the study of AAV latency; however, as more serotypes were discovered, its use became less frequent. In retrospect, AAV2 appears to be a tissue culture-adapted strain, and its primary receptor, heparan sulfate proteoglycan is not abundant on most target cells. This has prompted investigation into a wide-range rAAV serotypes for the efficacy and safety of in vivo AAT gene delivery resulting in various effective rAAV vectors for transgene delivery often specialized for the location of transduction. 3.3

4.

Preclinical and clinical development

Development of rAAV2-AAT In the late 1990s, investigation into rAAV2 as a gene therapy vector for the treatment of AAT was initiated [49]. Xiao et al. determined that hepatic delivery of rAAV2-AAT resulted in AAT production and secretion into the serum. The concept 4.1

that intramuscular (IM) delivery of rAAV2-AAT could be used to express high levels of AAT ectopically from myofibers was demonstrated in C57Bl6 and in severe combined immune deficiency (SCID) mice [50]. A single IM injection resulted in stable, therapeutically relevant serum AAT levels. It was hoped that increases in IM rAAV2-AAT doses would correspond to relative increases in serum AAT levels with the therapeutic significance being the avoidance of emphysema development without the necessity of regular protein replacement therapy. The original studies of IM administration of rAAV2-AAT were performed in both C57Bl6 and SCID mice in order to identify limitation due to the host immune response [50]. Surprisingly, different patterns of vector integration were observed in the two different strains [51]. Further study suggested that the host mutation resulting in immune deficiency, the deletion of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) was responsible for the difference in the molecular fate of rAAV genomes. Although AAT expression lasted up to 1 year with a single injection independent of DNA-PK activity, the presence of DNA-PK had a marked effect on the physical form of the persistent vector DNA. rAAV2-AAT genomes formed circular episomes in the presence of DNA-PK, with a predominance of singlecopy linear episomes and integrated vector genomes in its absence. The lack of formation of end-to-end junctions between rAAV2-AAT monomers fits well with the known role of DNA-PK in intermolecular recombination. Further investigation of DNA-PK’s involvement in rAAV2 transduction both in vitro and in vivo was executed to better understand the broader potential risk of rAAV2-mediated insertional mutagenesis [52]. DNA-PK was shown to facilitate episome formation and inhibit integration in cell-free systems, in tissue culture, and in liver (in addition to the earlier observations in muscle. Thus, a natural DNA repair mechanism in mammalian cells was found to greatly mitigate long-term risks of integration, mutagenesis and carcinogenesis. Further assessments of the safety and efficacy of IM rAAV2-AAT in animal models set the stage for the first human trial, which commenced in 2003 and was reported in 2006 [53,54]. In this first-in-human Phase I clinical trial of IM rAAV2-AAT in deficient adults, the rAAV2 vector capsid generated a humoral immune response, but no adverse effects presented, further supporting the safety of rAAV2 IM injection. In most patients receiving doses of 6.9  1012 vector genomes (vg) or higher, evidence of vector DNA spread into the peripheral blood appeared one to 3 days after injection. However, transgene expression was low (fourfold above background, 0.7% of threshold) and only transiently detectable indicating the necessity for higher vector doses to maintain consistent levels of expression. Development of rAAV1-AAT The potency of transgene expression observed with the rAAV2 vector stimulated evaluation into other serotypes for 4.2

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H. S. Loring & T. R. Flotte

the safety and efficacy of AAT delivery [55]. Lu et al. analyzed the murine IM transduction efficiency for five serotypes of rAAV (1, 2, 3, 4, 5). rAAV1 vector resulted in serum AAT levels 200-fold higher than rAAV2-AAT. It was hoped that therapeutic levels of serum AAT would be more readily achieved based on a switch from rAAV2 to rAAV1 capsid. The fact that this could be achieved by packaging an identical rAAV-AAT cassette (using the same promoters and AAV2derived ITR sequences cross-packaged into AAV1 capsid) allowed for maximal leverage of the existing preclinical and clinical safety data. Preclinical studies of rAAV1 to assess the safety for future translation into clinical treatments for AAT deficiency were performed in mice and New Zealand White rabbits [56]. The highest vector doses elicited an immune response in both mice and rabbit muscle tissue. Antibodies to both AAT and AAV1 were detected 1000-fold higher than control levels, diverging from the immunological findings associated with rAAV2. Murine and rabbit IM rAAV1-AAT delivery indicated dose-dependent AAT levels with significant vector infiltration mostly in the blood with observable vector concentrations in the peripheral organs and gonads at higher doses. The success associated with IM rAAV1-AAT delivery in animal models set the stage for another first-in-human Phase I clinical trial, this one for IM rAAV1-AAT [57]. Sustained levels of serum AAT were detected in the two highest dose cohorts of three (2.2  1013 and 6.0  1013 vg, respectively). The group that received the highest dose, 6.0  1013 vg, achieved serum AAT levels for at least a year despite positive T cell ELISPOT responses to AAV1 capsid epitope pools, indicating the persistence of transduced cells in spite of an effector T cell response. Although sustained AAT levels were achieved in this study, the levels attained were > 200-fold less than target therapeutic levels for preventing AAT lung disease. The need for even higher rAAV1-AAT doses necessitated a switch in vector production methodology from a doubleplasmid TFX method to a recombinant herpes simplex virus (HSV) complementation system [58,59]. The HSV-recombinant packaging system, when combined with efficient column chromatography for down-stream purification, enables the production of higher vector doses, but interestingly, also resulted in a more infectious vector preparation, which translated into a more potent product. Murine preclinical studies comparing the TFX and HSV-generated rAAV vectors indicated that for the same doses, HSV-produced vector resulted in higher serum AAT [59]. In humans, a linear relationship between the HSVproduced rAAV1-AAT dosage and AAT expression was found in all three dosage groups (6.0  1011, 1.9  1012, and 6.0  1012 vg/kg). Note that with the change to weight-based dosing the low-dose cohort here equates to the high-dose in the previous Phase I rAAV1-AAT IM study. Vector expression was sustained at levels ~ 3% of the therapeutic target [58]. Although the total vector genome amounts could be further increased, the concentration of vector could not be increased beyond ~ 5  1012 vg/ ml. This meant that increasing doses required increasing 4

volumes of vector injection. The top dosage group in the Phase II study required 100 IM injections of 1.35 ml each in order to deliver the rAAV1-AAT. Thus, further significant increases in the dose would not be feasible with this technique. Importantly, longer-term follow-up of the patients in the Phase II trial indicated that vector expression persisted for > 1 year at the 3% level, despite the presence of cellular infiltrates in the injected muscle and the presence of capsidspecific T cells in peripheral blood by g-interferon ELISPOT. This was observed in the absence of any immune suppressive medications [60]. In order to understand the mechanism of this persistence, Flotte et al. assessed the implications of the recruitment and proliferation of regulatory T cells on the long-term expression of AAT [60]. Tregs were found to account for 10% of infiltrating T cells within the injected muscle and correlated with the presence of Tregs with AAV1 capsid specificity in the peripheral blood. These findings bode well for the prospect that further dosage increases of muscle-delivered rAAV1-AAT could result in a sustained therapeutic response. Given the physical limitation of IM delivery described above, however, this is being pursued using an isolated limb infusion (ILI) method. The ILI method will allow for a substantially greater volume of vector to be delivered to the muscle, potentially increasing the sustained levels from the current 3 -- 100% of the target (11 µM, 570 µg/ ml, or 57 mg/dl). The potential advantage of progressing to a Phase IIb/III trial of rAAV1-AAT muscle delivery via ILI is that it would presumably elicit a similar Treg response as has been seen in the IM trials. In contrast it is not clear whether protective Treg responses would be elicited after systemic injection with resultant delivery of vector to the liver. An effector T cell response after liver delivery could result in transaminase elevations as has been seen in the hemophilia B trials [61], which might be exacerbated by the hepatocyte load of Z-mutant AAT [62]. The long-term persistence of rAAV-mediated gene transfer could be of particular importance when considering the implication of the humoral immune response to rAAV. The fact that anti-AAV capsid neutralizing antibodies have been observed in rAAV1-AAT IM trials and most other rAAV human trials implies that the efficacy of repeated delivery is likely to be impaired. The rationale for current studies depends, therefore, on the concept that expression will be persistent for years after a single injection of vector.

Development of other rAAV serotypes and other routes of delivery

4.3

Various studies have implemented AAV serotype 2 and 1 intramuscularly and intravenously; however, its inefficiency requires high vector doses to adequately target the lungs [63]. By comparing AAV5 and AAV2 vectors, Crystal et al. found that AAT delivered by AAV5 was five times higher than that delivered by AAV2 injected via either the intrapleural or IM routes. Intrapleural administration of AAV5 resulted in ten

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Current status of gene therapy for a-1 antitrypsin deficiency

times higher serum AAT levels than IM administration of the same vector [64]. Although the AAV5 vector was successful in providing the lungs with AAT, Crystal et al. aimed to analyze whether a more efficient AAV vector existed that would also be capable of circumventing the host immune system [65]. AAT delivery was analyzed for 25 AAV vectors administered intrapleurally. The rhesus AAV rh.10 serotype yielded the highest levels of AAT expression, sustaining > 2.5 times the therapeutic level in humans; moreover, the AAVrh.10 vector was capable of increasing AAT levels in spite of the immune response generated by AAV2 and AAV5. Recent progress on intrapleural AAT gene therapy has included non-human primate preclinical studies in anticipation of first-in-human trials. As lung pathology is the primary life-limiting concern in most AAT-deficient patients, multiple rAAV serotypes and two methods for vector introduction were analyzed in mice for the efficacy concerning AAT delivery to the lungs [66]. To ascertain the effect of the invasive delivery of rAAV vector via the traditional intratracheal route on vector transduction and expression efficiencies it was compared with noninvasive delivery intranasally. For the majority of rAAV vectors analyzed, serum AAT levels were two to threefold higher by intratracheal delivery of the transgene. The highest AAT expression in the bronchoalveolar lavage fluid and lung tissue resulted from the rAAV serotype 8 vector followed by serotypes 9 and 5, independent of the mode of delivery. Intratracheal delivery of rAAV8 harbors potential for future applications in pulmonary gene transfer. One important caveat to studies of alternative serotypes for AAV delivery to lung is that the tropism of vectors for mouse lung is so divergent from that seen in humans. A study by Flotte et al. [67] using a dual reporter luciferase system demonstrated that the relative tropism of rAAV1 as compared with rAAV5 was much better predicted by chimpanzees, the closest evolutionary relative of humans than even by smaller primates. In that study rAAV1 was found to be the more potent and less immunogenic capsid for lung delivery. A consideration of airway surface delivery would not be complete without consideration of an intratracheally lentivirus-based approach to AAT gene therapy described by Wilson et al., from the Kotton laboratory [68]. In that study, mice treated with a lentiviral vectors expressing AAT from the EF-1a promoter expressed AAT long-term primarily by transduction of alveolar macrophages and were rendered resistant to elastase-induced emphysema. Development of vectors for AAT liver disease Gene therapy for AAT liver disease diverges in a fundamental way from gene therapy for the lung disease. Although the lung disease is due to the lack of AAT’s antiprotease activity, AAT liver disease is due to the toxic gain of function of the common Z-mutant (E342K) form of AAT within hepatocytes, where it misfolds and forms polymers, leading to impaired secretion, ER dilation, and large aggregates [13,22]. Thus, strategies to treat 4.4

AAT-deficient-liver disease focus on either down-regulating endogenous Z-mutant AAT expression or ameloriating the effects of Z-AAT on hepatocytes by facilitating proper folding or secretion. Recently, a single dual function rAAV vector was injected into mice in an attempt to correct both the degradation of the lungs caused by lower concentrations of serum AAT and the toxicity caused by buildup of mutant AAT in the liver [69]. Incorporation of specific miRNA sequences along with AAT gene into rAAV9 targeted the mRNA for the mutant ZAAT in the liver while also allowing for the production of serum M-AAT, resistant to miRNA knockdown. Implementing RNA interference, endogenous Z-AAT was reduced within the hepatocytes along with evidence of polymerization and inflammation. By also packaging an miRNA-resistant AAT gene within the vector, serum M-AAT increased whereas toxicity within the liver decreased. Markedly, a little effect was exhibited on endogenous miRNAs yielding insight into future applications of dual function vectors for the treatment of other diseases as well. 5.

Conclusion

Gene therapy for lung disease due to AAT deficiency has progressed to Phase II clinical trials of IM delivery of rAAV1-AAT, and additional serotypes and routes of delivery of rAAV-AAT have demonstrated preclinical proof-of-principle. The muscle-based rAAV-AAT approach is progressing to a Phase IIb/III trial, moving to the ILI method to make the delivery of larger volumes more feasible. The presence of an active Treg response to capsid seems to be enabling longterm persistence of AAT delivery even in the face of effector T cell responses to capsid [60]. It remains to be seen whether such a Treg response would be found with other routes of delivery. The feasibility of other routes of rAAV-AAT delivery has also been demonstrated. Intrapleural administration has been shown to produce a particularly robust response, but lung and liver delivery for augmentation also could be pursued. The likelihood of gene therapy to reduce the pathology due to AAT liver disease is more complex, but initial results with a bifunctional vector also demonstrated an important proof-of-principle [69]. 6.

Expert opinion

The current status of AAT gene therapy must be considered in the broader context of the field. The first approval of a human gene therapy product in the Western world occurred in November 2012, when the EMA approved Glybera, a gene therapy for lipoprotein lipase deficiency [70]. The fact Glybera consists of an rAAV1 vector delivered by IM injection and that muscle-based rAAV1 delivery has advanced further than any another modality for gene therapy of AAT lung disease seems to bode particularly well for the success of rAAV1-AAT by ILI. The presence of capsid-specific Tregs may well

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ultimately prove to be of pivotal importance to the success of such an approach. Countering that enthusiasm, it should be noted that the since Glybera has not yet been marketed commercially and the costs of good may be high enough to present an obstacle to its use. Longer term, the emergence of new genome editing technologies based on the CRISPR/Cas9 system would seem to open up the possibility of a truly definitive therapy. The efficiency of site-specific nicking or double-strand breakage with extremely high fidelity would seem to offer great promise for correction of both the liver and lung disease due to the ZAAT mutation. The unusual genetic homogeneity of AAT deficiency would make a correction of Z-AAT applicable to > 90% of affected patients. The mode of delivery for a genome editing approach may not be obvious. The availability of techniques to reprogram somatic cells, producing induce pluripotent stem (iPS) cells, might make possible an autologous cell transplant approach. In such an approach, a Z-AAT patient’s IPS cells derived from skin or peripheral blood might be subjected to editing with Cas9 directed appropriate guide RNAs (gRNAs) with specificity to the mutation site. Ideally, two double-strand breaks flank the mutation site, and a donor DNA (dDNA) with the corresponding wild-type sequence is also delivered. Once an iPS cell clone is edited, and the correction is confirmed, corrected cells could then be transplanted to the patient. With sufficient engraftment, this approach might be able to correct all aspects of the disease. The use of iPS cells with an otherwise identical genetic identity to accomplish this correction might avoid the need for immune suppression Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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that is inherent in current allogeneic organ and cell transplantation. Perhaps the ideal form of definitive correction might occur if all components required for genome editing could be delivered to the liver of Z-AAT patients in situ. This would include a gene encoding the Cas9 recombinase, the appropriate gRNAs for the sites flanking the mutation site, and the dDNA with the corrected sequence. The existing rAAV serotype vectors may be efficient enough for delivery of each of these components to a majority of hepatocytes. If corrected hepatocytes hold any form of selective advantage over those expressing Z-mutant AAT, this could represent the proverbial ‘silver bullet’ of therapy for these patients. Although many questions remain about the potential for adverse effects from any off-target hits from the CRISPR/Cas9 system, the prospect now exists for a truly personalized, definitive correction of this life-limiting genetic disorder.

Declaration of interest T Flotte was a founder of Applied Genetic Technologies Corporation (NASDAQ:AGTC) but donated founder equity to the Alpha One Foundation, who subsequently transferred it to AlphaNet. He also serves as a Scientific Advisor to Dimension Therapeutics (in which he holds equity) and Editas Medicine, and may be entitled to royalties as an inventor of some technology described here. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Affiliation

Heather S Loring & Terence R Flotte† Author for correspondence University of Massachusetts Medical School, Gene Therapy Center, Department of Pediatrics, Suite 340 55 Lake Avenue North Worcester, MA 01655, USA Tel: +1 508 856 2107; Fax: +1 508 856 8181; E-mail: [email protected]

Current status of gene therapy for α-1 antitrypsin deficiency.

As a common monogenic disease, α-1 antitrypsin (AAT) deficiency has undergone thorough investigation for the development of gene therapy. The most com...
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