Alpha-1 antitrypsin deficiency (AATD) is a common genetic disorder that can lead to both liver and lung disease and currently affects an estimated 3.4 million patients worldwide. Alpha-1 antitrypsin (AAT) is encoded by SERPINA1 and is primarily secreted by hepatocytes making it the most abundant serum antiprotease. One of the most common disease variants in AATD is a mutation resulting in a glutamate to lysine (Glu342Lys) substitution known as the PiZ allele or Z-AAT. In contrast to the normal PiM allele (M-AAT), the Z-AAT protein is prone to polymerization and consequently is either directed for proteolysis or aggregates in the endoplasmic reticulum of hepatocytes. With up to 85% of the AAT protein being retained as polymers or degraded in the liver, it sets the stage for both the loss-of-function (lung) and toxic gain-of-function (liver) diseases observed in AATD patients. Normally AAT is secreted and diffuses throughout the bodily organs where it protects tissue from the `unchecked' or `off-target' activity of proteases. In the lungs, the loss-of-function phenotype is due to the imbalance of protease/antiprotease homeostasis. Specifically, the protease known as neutrophil elastase, which is secreted by neutrophils as a form of innate immunity, goes unchecked and over years leads to the degradation of the lung architecture. This eventually manifests as chronic obstructive pulmonary disease (COPD) and emphysema. In contrast, Z-AAT aggregation and polymerization causes liver disease by a toxic gain-of-function mechanism due to accumulation of misfolded protein in the hepatocytes whereby 10-20% of PiZ homozygote patients suffer from clinical liver disease ranging from fulminant liver failure and cirrhosis to hepatocellular carcinoma. Our group has developed strategies for simultaneous gene augmentation with mutant gene reduction for both lung and liver disease with dual function vectors, but an unmet need remains. We need to address liver disease in a young, actively dividing liver as is the case of AATD liver disease in the pediatric population. Furthermore, gene editing approaches may offer longer-term solutions over episomal AAV gene therapy for adult livers that are slowly turning over due to disease. There are two notable advancements that will support the development of this second generation of rAAV-based therapies for liver disease. The first of these advances is that we now appreciate that homologous recombination (HR) with AAV can be achieved at high enough efficiency without the use of nuclease to have a meaningful clinical impact for liver disorders. The second important development in the AAV-mediated gene editing field is the realization that certain AAV serotypes are better at achieving nuclease-free homologous recombination. Recently new AAV members of clade f known as AAV-HSCs were isolated form hematopoietic stem cells, and these vectors have shown high HR activity. Thus, this grant aims to develop novel nuclease- free gene-editing strategies that will address the permanent correction of AATD by using AAV-mediated homologous recombination into either the Albumin or Serpina1 locus. We hypothesize that nuclease-free AAV- mediated gene editing is feasible, efficient and a safe therapeutic approach for treating alpha-1 antitrypsin deficiency. We will test this hypothesis with the following three aims.
In Aim 1 we will use mice with human liver xenografts to identity rAAV-HSC capsids that have a human hepatocyte tropism and increased homologous recombination activity.
In Aim 2 we will compare nuclease-free AAV-mediated, gene editing with AAV3b at either the Albumin or SerpinA1 locus in a non-human primate model. Finally in Aim 3 we will do a head-to- head comparison of the optimal AAV-HSC capsid identified in Aim1 against AAV3b at the more efficient locus identified in Aim 2. Overall, the grant aims to determine the ideal locus and best capsid with which to achieve AAV? mediated, nuclease-free gene editing for alpha-1 antitrypsin deficiency. It should be noted that the results of this research would also directly impact the CRISPR/Cas9 and ZFN fields broadly, as currently the most efficient way of delivering DNA templates for HDR with the nuclease-dependent gene editing approaches still relies on rAAV. Thus, we feel that our data will serve as a HDR benchmark from which the nuclease- dependent approaches can improve upon by incorporating DNA breaks and nicks into the approach.

Public Health Relevance

Alpha 1-antitrypsin deficiency (?1-antitrypsin deficiency, AATD) is a genetic disorder that causes defective production of alpha 1-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. In patients with two copies, this mutation leads to severe deficiency of AAT and can result in liver disease in some children and adults, which is due to the toxicity of the Z-AAT mutant protein. Our laboratory is exploring therapies to address this disease by using viral capsids to deliver a `corrective' DNA payload. These capsids when injected into circulation can hone to the liver where the infect hepatocytes and deliver the `corrective' payload to the cells. The DNA payload will then will insert itself into the genetic lesion of sick hepatocytes and correct the disease at the genetic level.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Doo, Edward
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University of Massachusetts Medical School Worcester
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Borel, Florie; Sun, Huaming; Zieger, Marina et al. (2018) Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema. Proc Natl Acad Sci U S A 115:2788-2793
Mueller, Christian; Gernoux, Gwladys; Gruntman, Alisha M et al. (2017) 5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency. Mol Ther 25:1387-1394
Gernoux, Gwladys; Wilson, James M; Mueller, Christian (2017) Regulatory and Exhausted T Cell Responses to AAV Capsid. Hum Gene Ther 28:338-349
Borel, Florie; Tang, Qiushi; Gernoux, Gwladys et al. (2017) Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of ?-1 Antitrypsin Deficiency. Mol Ther 25:2477-2489
Loring, Heather S; ElMallah, Mai K; Flotte, Terence R (2016) Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans. Hum Gene Ther Methods 27:49-58
Gruntman, Alisha M; Su, Lin; Su, Qin et al. (2015) Stability and compatibility of recombinant adeno-associated virus under conditions commonly encountered in human gene therapy trials. Hum Gene Ther Methods 26:71-6
Borel, Florie; Kay, Mark A; Mueller, Christian (2014) Recombinant AAV as a platform for translating the therapeutic potential of RNA interference. Mol Ther 22:692-701
Flotte, Terence R; Mueller, Christian (2014) What is suppression of anti-adeno-associated virus capsid T-cells achieving? Hum Gene Ther 25:178-9
Kiem, Hans-Peter; Baum, Christopher; Bushman, Frederic D et al. (2014) Charting a clear path: the ASGCT Standardized Pathways Conference. Mol Ther 22:1235-1238