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-edit...