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α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR‐34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser(574). Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is α-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a–e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research.

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology., Borel et al. describe two studies based on misfolded human α-1 antitrypsin (A1AT). First, when A1AT is expressed in livers of NSG, mice it allows for reproducible engraftment of human hepatocytes. Second, gene editing of hepatocytes to decrease misfolded protein results in expansion of corrected cells and amelioration of liver disease.

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%–3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%–2.5% of the target level from years 1–5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated., Alpha-1 antitrypsin deficient patients injected intramuscularly with a rAAV1 vector demonstrated 5 years of stable transgene expression after a single dose, coincident with an anti-capsid Treg response and exhaustion of CD8 cells. Mueller et al. also observed partial correction of disease biomarkers despite serum levels below the target level for protein replacement.

Adeno-associated viral vectors have emerged as an important tool for human gene therapy, having demonstrated high transduction efficiency in a broad range of target tissues, a good safety profile in animal models and human clinical trials, and prospective long-lasting gene expression. First discovered 20 years ago, RNA interference (RNAi) has become another important tool for human gene therapy, enabling scientists to move on from classical gene transfer to gene silencing approaches, or combinations thereof. In this chapter, we describe a simple step-by-step method that will allow gene silencing novices to design their own artificial miRNAs against a target of their choice, clone these miRNAs into an AAV-based vector, and rapidly screen for highly efficient artificial miRNAs. The described method takes into consideration recent advances in the field including miRNA processing from various cellular miRNA backbones, choice between polymerase II and III promoters, and the potential impact of these factors on toxicity as it relates to off-targeting and to saturation of the endogenous RNAi machinery.

This protocol describes a method of delivering adeno-associated viral (AAV) vectors to the intrathecal space of nonhuman primates for CNS-directed gene therapy. It includes the surgical implantation of the catheter, vector infusion, necropsy, laser-capture microdissection of motor neurons, and gene expression analysis. This method allows efficient and reproducible delivery, and would be of interest to test gene therapy vectors for the treatment of disorders of the central nervous system of nonhuman primates. This protocol was tested in cynomolgus macaques and may be adapted for AAV delivery to different species of large animals.

Adeno-associated viral vectors have emerged as an important tool for human gene therapy, having demonstrated high transduction efficiency in a broad range of target tissues, a good safety profile in animal models and human clinical trials, and prospective long-lasting gene expression. First discovered 20 years ago, RNA interference (RNAi) has become another important tool for human gene therapy, enabling scientists to move on from classical gene transfer to gene silencing approaches, or combinations thereof. In this chapter, we describe a simple step-by-step method that will allow gene silencing novices to design their own artificial miRNAs against a target of their choice, clone these miRNAs into an AAV-based vector, and rapidly screen for highly efficient artificial miRNAs. The described method takes into consideration recent advances in the field including miRNA processing from various cellular miRNA backbones, choice between polymerase II and III promoters, and the potential impact of these factors on toxicity as it relates to off-targeting and to saturation of the endogenous RNAi machinery.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease; survival in ALS is typically 3-5 years. No treatment extends patient survival by more than three months. Approximately 20% of familial ALS and 1-3% of sporadic ALS patients carry a mutation in the gene encoding superoxide dismutase 1 (SOD1). In a transgenic ALS mouse model expressing the mutant SOD1(G93A) protein, silencing the SOD1 gene prolongs survival. One study reports a therapeutic effect of silencing the SOD1 gene in systemically treated adult ALS mice; this was achieved with a short hairpin RNA, a silencing molecule that has raised multiple safety concerns, and recombinant adeno-associated virus (rAAV) 9. We report here a silencing method based on an artificial microRNA termed miR-SOD1 systemically delivered using adeno-associated virus rAAVrh10, a serotype with a demonstrated safety profile in CNS clinical trials. Silencing of SOD1 in adult SOD1(G93A) transgenic mice with this construct profoundly delayed both disease onset and death in the SOD1(G93A) mice, and significantly preserved muscle strength and motor and respiratory functions. We also document that intrathecal delivery of the same rAAVrh10-miR-SOD1 in nonhuman primates significantly and safely silences SOD1 in lower motor neurons. This study supports the view that rAAVrh10-miR-SOD1 merits further development for the treatment of SOD1-linked ALS in humans.

Significance Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is a genetic disease, α-1 antitrypsin (AAT) deficiency. Humans have only one gene that codes for the AAT protein, but mice have up to six, which made it impossible for decades to create a mouse model of the disease. Here we succeeded in creating this mouse model using CRISPR technology to target all of the mouse genes at once. Importantly, this mouse model spontaneously develops lung disease and recapitulates many aspects of the human disease. We anticipate that this model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research., Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is α-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a–e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease caused by degeneration of motor neurons leading to rapidly progressive paralysis. About 10% of cases are caused by gain-of-function mutations that are transmitted as dominant traits. A potential therapy for these cases is to suppress the expression of the mutant gene. Here, we investigated silencing of SOD1, a gene commonly mutated in familial ALS, using an adeno-associated virus (AAV) encoding an artificial microRNA (miRNA) that targeted SOD1 In a superoxide dismutase 1 (SOD1)-mediated mouse model of ALS, we have previously demonstrated that SOD1 silencing delayed disease onset, increased survival time, and reduced muscle loss and motor and respiratory impairments. Here, we describe the preclinical characterization of this approach in cynomolgus macaques (Macaca fascicularis) using an AAV serotype for delivery that has been shown to be safe in clinical trials. We optimized AAV delivery to the spinal cord by preimplantation of a catheter and placement of the subject with head down at 30° during intrathecal infusion. We compared different promoters for the expression of artificial miRNAs directed against mutant SOD1 Results demonstrated efficient delivery and effective silencing of the SOD1 gene in motor neurons. These results support the notion that gene therapy with an artificial miRNA targeting SOD1 is safe and merits further development for the treatment of mutant SOD1-linked ALS.

This protocol describes an enzyme-linked immunosorbent assay (ELISA) to specifically detect Z-alpha-1 antitrypsin (AAT), the most common protein variant associated with alpha-1 antitrypsin deficiency. This "sandwich" ELISA relies on an anti-Z-AAT specific capture antibody and a HRP-conjugated anti-AAT detection antibody. This method would be of interest to identify and quantify Z-AAT in a variety of samples such as cell culture medium, cell or tissue lysate, animal or patient serum. Because this method is specific and sensitive, it would be particularly valuable for detection of Z-AAT in the presence of background M-AAT, for instance when quantifying silencing of Z-AAT in patients undergoing M-AAT augmentation therapy.

This protocol describes the design, cloning, and in vitro screening of artificial microRNAs (miRNAs) to silence alpha-1 antitrypsin (AAT). This method would be of interest to silence AAT in a variety of in vitro or in vivo models, and prevalidated sequences against human AAT are provided. This simple 5-day protocol may more generally be used to design artificial miRNAs against any transcript.

RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed.

Rationale: The most prevalent genetic cause of chronic obstructive pulmonary disease is alpha-1 antitrypsin (A1AT) deficiency, a disorder that has yet to be widely modeled in animals because of species-specific differences between rodents and humans. Objectives: To address these challenges, we engineered two A1AT ferret models using zygote gene editing to test the hypothesis that unopposed protease activity within the lung leads to emphysema and bronchitis. Methods: Guide RNAs targeting exon 2 (for knockout) and exon 5 (for Z-allele mutation, Pi*Z) of the ferret A1AT gene were injected into ferret zygotes with Cas9 mRNA. For PI*Z targeting, a short oligonucleotide carrying the mutation was included. Offspring were genotyped and plasma levels of A1AT determined by Western blot before entry into a longitudinal study. Adult A1AT ferrets underwent bronchoscopy and FlexiVent over time to characterize lung disease. Control animals were wild-type (WT) and age, sex, and size matched. Results: A1AT-deficient ferrets spontaneously develop hallmarks of emphysema as early as 3 months when compared with matched WT control ferrets. Over time, A1AT-KO ferrets reached an inspiratory capacity of 117% ± 5% (WT, 57.4 ml; A1AT, 67.5 ml) and compliance of 111% ± 4% (WT, 5.5 ml/cm H(2)O; A1AT, 6.1 ml/cm H(2)O; N = 8 pairs A1AT/WT, P

This protocol describes the design, cloning, and in vitro screening of artificial microRNAs (miRNAs) to silence alpha-1 antitrypsin (AAT). This method would be of interest to silence AAT in a variety of in vitro or in vivo models, and prevalidated sequences against human AAT are provided. This simple 5-day protocol may more generally be used to design artificial miRNAs against any transcript.

This protocol describes an enzyme-linked immunosorbent assay (ELISA) to specifically detect Z-alpha-1 antitrypsin (AAT), the most common protein variant associated with alpha-1 antitrypsin deficiency. This "sandwich" ELISA relies on an anti-Z-AAT specific capture antibody and a HRP-conjugated anti-AAT detection antibody. This method would be of interest to identify and quantify Z-AAT in a variety of samples such as cell culture medium, cell or tissue lysate, animal or patient serum. Because this method is specific and sensitive, it would be particularly valuable for detection of Z-AAT in the presence of background M-AAT, for instance when quantifying silencing of Z-AAT in patients undergoing M-AAT augmentation therapy.

Huntington's disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken β-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CβA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CβA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CβA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CβA promoter can provide an effective and safe dose of a human huntingtin miRNA.

MicroRNAs (miRNAs) are evolutionary conserved small non-coding RNAs that regulate gene expression by mediating post-transcriptional silencing of target genes. Since miRNAs are involved in fine-tuning of physiological responses, they have become of interest for diagnosis and therapy of a number of diseases. Moreover, the role of dysregulated miRNAs in maintaining the malignant phenotype has profound implications for cancer therapy. We will review the best defined cellular miRNAs and changes in their expression profile in hepatocellular carcinoma (HCC). Cellular miRNAs can also be released into the circulation, and these miRNAs are detected in most body fluids. Circulating miRNAs are associated with HCC and are possible biomarkers. Finally, by affecting several clinically relevant targets, artificially increasing or decreasing the expression level of a given miRNA offers fascinating therapeutic perspectives. We will therefore highlight recent developments in miRNA-based gene therapy with a focus on their therapeutic potential for HCC. (C) 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Correspondance: Bruno.Touraine@univ-montp2.fr; International audience; 1-Amino cyclopropane-1-carboxylic acid deaminase (AcdS) is an enzyme that degrades the precursor of plant hormone ethylene. AcdS activity has been identified in many soil bacteria. It has been proposed to play an important role in plant-growth promotion by rhizobacteria. It would lower ethylene level via uncharacterized signaling pathways in the host plant. To further investigate the role of AcdS and the involvement of ethylene signaling pathway in plant development responses to rhizobacteria, we used the model plant Arabidopsis thaliana. We compared the changes in root architecture and root hair length induced by four rhizobacteria (Phyllobacterium brassicacearum STM196, Pseudomonas putida UW4, Rhizobium leguminosarum 6v. viciae 128C53K, Mesorhizobium loti MAFF303099) and by their respective acdS-deficient mutants. All the mutant strains induced similar changes in lateral root development as their WT counterparts. By contrast, root hairs of seedlings inoculated with the acdS mutant strains were significantly longer than those of the plants inoculated with the WT strains. Overall, our results would suggest that rhizobacterial AcdS activity affects local regulatory mechanisms in plant roots, and not lateral root development that is under systemic regulation involving shoot-root dialog

Immune responses against recombinant AAV (rAAV) capsid are a major concern in clinical trials. It was first described in hemophilic patients injected by intrahepatic delivery that AAV capsids elicited a cytotoxic lymphocyte response leading to clearance of transduced hepatocytes. On the other hand, intramuscular delivery of rAAV1 in humans induced a tolerogenic response against rAAV capsid mediated by regulatory T cells leading to stable expression in patients for more than 3 years. Several parameters could modulate this response and among them the mode of delivery. While transfer to the intact CNS has been shown to elicit a minimal T cell-mediated response without a salient plasma cell-mediated immune response in preclinical animal studies, these data are lacking in primate studies after intrathecal (IT) injection. In our study, African Green primates were injected intrathecally with a serotype 10 AAV vector (AAVrh10), described to transduce efficiently the central nervous system (CNS), expressing artificial miRNAs against superoxide dismutase 1 (SOD1) as a therapy for familial amyotrophic lateral sclerosis (ALS). This vector was administered at a dose of 6e12 viral genomes per kilogram modeled directly off the anticipated dose per patient in the clinical trial and based on IT rAAV delivery in primate studies showing robust cortical and spinal cord transduction. We are currently characterizing what type of immune response is elicited against the AAV capsid after IT delivery. A particular interest will be given to cytokine secretion mediated by restimulated T lymphocytes in vitro and to neutralizing antibodies directed against AAVrh10 and their potential cross-reactivity to other serotypes. In addition to the proof-of-concept of knockdown and spread of AAV vector after delivery to the CNS of primates, this study is important for the safety of IT delivery in large animal models before translation to human clinical trials.

Constitutive expression of short hairpin RNAs (shRNAs) may cause cellular toxicity in vivo and using microRNA (miRNA) scaffolds can circumvent this problem. Previously, we have shown that embedding small interfering RNA sequences targeting apolipoprotein B100 (ApoB) in shRNA (shApoB) or miRNA (miApoB) scaffolds resulted in differential processing and long-term efficacy in vivo. Here we show that adeno-associated virus (AAV)-shApoB- or AAV-miApoB-mediated ApoB knockdown induced differential liver morphology and transcriptome expression changes. Our analyses indicate that ApoB knockdown with both shApoB and miApoB resulted in alterations of genes involved in lipid metabolism. In addition, in AAV-shApoB-injected animals, genes involved in immune system activation or cell growth and death were affected, which was associated with increased hepatocyte proliferation. Subsequently, in AAV-miApoB-injected animals, changes of genes involved in oxidoreductase activity, oxidative phosphorylation and nucleic bases biosynthetic processes were observed. Our results demonstrate that long-term knockdown of ApoB in vivo by shApoB or miApoB induces several transcriptome changes in murine liver. The increased hepatocyte profileration by AAV-shRNA may have severe long-term effects indicating that AAV-mediated RNA interference therapy using artificial miRNA may be a safer approach for familial hypercholesterolemia therapy.

Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5' and 3' cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 10(11) genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.

Alpha-one antitrypsin (AAT) deficiency is a common autosomal co-dominant genetic disorder. This condition affects 1:2500 individuals of European ancestry, leading to the development of lung and liver disease. Within North American and Northern European populations, an estimated 4% of individuals are carriers of mutant alleles. AAT deficiency presents with an emphysema phenotype in the lungs of older subjects. AAT deficient subjects can also suffer from liver disease of varying severity; however, lung disease is the principle cause of death. AAT is a protease inhibitor predominantly synthesized in the liver that belongs to the serine protease inhibitor (serpin) family. Upon secretion into the blood stream, AAT enters the lungs where it inactivates excess neutrophil elastase, thereby preventing damage to the alveoli. Mutations of the Serpina1 gene can lead to reduced serum levels of AAT and decreased protein functionality, allowing for unrestricted elastin breakdown, pulmonary inflammation and eventual emphysema. Currently, an animal model simulating the lung condition does not exist, which severely limits the development of therapeutics. This is due to the higher genomic complexity of mice compared to humans. Indeed due to amplification events, C57BL/6 mice have five genes that are homologous to human SERPINA1. To address this we generated a quintuple gene knockout using CRISPR/Cas9 system via zygote microinjection. We generated three founding lines in which all 5 copies of the gene were disrupted. Mice from all three lines demonstrate absence of hepatic and circulatory AAT protein as well as a reduced capability to inactivate neutrophil elastase. We also characterized the lung phenotype in response to a lipopolysaccharide challenge, where the model recapitulated many characteristics of the human lung disease including decreased elastance and increased compliance, and lung morphometry was also affected. Genomic and transcriptomic characterization will be presented. Future work will include challenges with cigarette smoke, a well-known disease accelerator in patients. Further, the ongoing generation of a new transgenic model carrying both the quintuple disruption of the murine Serpina1 genes and a single copy of the Z variant of human SERPINA1 will bring to the field the ultimate disease model that will finally allow researchers to evaluate the effects of liver-directed gene augmentation in the presence of Z-AAT polymers.

Intrathecal (IT) delivery of adeno-associated viral (AAV) vector is currently used for the treatment of neurodegenerative disorders. To date, there is no standardized method to deliver the vector into the cerebrospinal fluid (CSF). Several parameters could impact the transduction efficacy among them the dose, the AAV serotype or the volume of injection. In this study, we analyzed the impact of large vector volume administered in the lumbar CSF on the vector biodistribution. Cynomolgus monkeys were injected with a serotype 10 AAV vector (AAVrh10) a serotype known to transduce efficiently the CNS via a pre-implanted catheter. The catheter was inserted after a hemilaminectomy at approximately the L5 vertebra, and advanced intrathecally with the tip located near the thoracolumbar junction. The rAAVrh10 (5.0 ml) was administered as two 2.5 ml infusions (the second infusion was approximately six hours after initiation of the first infusion). The infusion rate was 7.5 ml/hr over approximately 20 minutes. For the duration of both infusion, the NHPs were restrained in a prone position with the restraint table tilted approximately 30 degrees head-down. The vector biodistribution was analyzed by digital PCR and the results demonstrate that viral genomes are detected along the entire length of the spinal cord but also in substantial levels in peripheral organs. Due to viral genomes detected in lymphoid organs, we are currently analyzing the immune response to AAV capsid. These results are important for assessing the safety of IT delivery in large animal models before translation to human clinical trials.

Background Controlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds. Results Highly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA. Conclusion Our results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.

Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.

Alpha-one antitrypsin deficiency (AATD) is a genetic disorder that causes defective production of alpha-one antitrypsin (AAT), leading to decreased AAT activity in the blood and lungs, and deposition of excessive mutant Z-AAT protein in liver cells. There are several forms and degrees of deficiency, principally depending on whether the patient has one or two copies of the affected gene. Approximately 4% of the North American and Northern European populations possess at least one copy of a mutant allele.The current standard of care is protein replacement by frequent infusions, but gene replacement therapy could provide long-term correction of the serum levels after a one-time treatment only, which would offer a significant improvement in the quality of life of the patients. Towards this goal, a recent clinical trial tested injecting recombinant adeno-associated virus serotype 1 expressing M-AAT intramuscularly. However the therapeutic AAT serum threshold of 11μμM was not reached. Future efforts to achieve therapeutic levels will revolve around dose escalation and more efficient physical delivery.While the next trial may achieve therapeutic levels with the current AAV1-CB-AAT vector, it is worth further optimizing as it may result in a more efficient vector requiring a lower dose to achieve therapeutic levels. Achieving therapy with a lower dose has substantial benefits in the long run in terms of cost of production and more importantly potential immune responses to capsid. The current AAV1-CB-AAT gene therapy vector will be optimized through four independent strategies hypothetically leading to an increase of alpha-one antitrypsin serum levels in mice: 1) replacement of the secretion signal by secretion signals from other human secreted proteins, 2) modification of the AAV capsid either by triple tyrosine mutation or DNA shuffling, 3) optimization of the transgene to create self-complementary vectors with a codon-optimized AAT sequence, 4) addition of a hepatocyte-specific cis-regulatory module allowing recruitment of liver-specific transcription factors.In vivo data will be presented for each strategy. Future work will combine the validated candidates into a single vector for further evaluation; the use of this optimized vector may translate into a therapeutic effect for the AATD patients.

Alpha-1 antitrypsin (AAT) deficiency is a common autosomal co-dominant genetic disorder. This condition affects up to 1:2500 individuals of European ancestry, leading to the development of lung and in certain cases liver disease. AAT, as a member of the serpin family, is a protease inhibitor predominantly synthesized in the liver. Upon secretion into the blood stream, AAT's principle physiological role occurs in the lungs where it inactivates excess neutrophil elastase, thereby preventing potential damage to the alveoli. In humans, a glutamate to lysine change at position 342 of the protein, known as a PiZ mutation, accounts for over 90% of cases. This and other mutations lead to reduced serum levels of AAT in addition to reduced protein function. With time, the decreased levels of AAT allows for unopposed neutrophil elastase activity, causing the eventual breakdown of alveolar elastin thus resulting in emphysema. Currently, a serum deficient genetic animal model simulating the lung condition does not exist, which severely limits the development of innovative therapeutics.Our goal is to use CRISPR/Cas9 technology to generate an AAT knock-out mouse to study the pulmonary disease. Unlike humans, mice have five highly conserved tandem copies of the serpinA1 gene. This phenomenon has made it difficult to create knockout mice using traditional approaches. With the advent of novel genome editing techniques, this problem has reached a feasible threshold. To this end, we designed four guide RNA (gRNA) constructs to target conserved regions within all 5 copies of the serpinA genes. The gRNA constructs were initially evaluated using a single strand annealing assay. By co-transfecting a gRNA/Cas9 complex with appropriate reporters, the intensity of resulting GFP measured target efficiency. Afterwards, embryonic stem cell transfection and zygote microinjection were used to deliver the genome editing complex for in vitro and in vivo studies.During the screening process, 200 embryonic stem cell samples and 40 transgenic mice were evaluated by quantifying AAT serum levels and sequencing specific target regions within the genome. Upon completion, several knock-outs were identified and founder lines were established.Ongoing studies aim to determine the susceptibility of developing emphysema after challenging the knockout mice with porcine pancreatic elastase. We expect the mice will display a distinguishable pulmonary phenotype, aside from decreased levels of AAT as determined by ELISA, which will allow for the characterization of airway pathophysiology. This mouse model is of particular clinical significance as it presents the first opportunity to test current and novel therapeutics aimed at augmenting serum levels of AAT.

ABC transporters export clinically-relevant drugs and their over-expression causes multidrug resistance. In order to knock-down ABC transporters, ABCC1 and ABCC2, 13 shRNAs were developed. Four shRNA candidates were tested in vivo using self-complementary adeno-associated virus serotype 8. A strong, specific knock-down of Abbc2 was observed in mice liver, but at the cost of toxicity caused by oversaturation of the RNAi machinery due to high shRNA expression. Subsequent generation of artificial miRNAs showed better efficacy profile. These results demonstrate the feasibility of knocking down Abbc2 via AAV-delivered shRNAs to the liver, and encourage the use of miRNA in further therapeutics development.