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The complex mechanisms underlying chronic pain and the challenges of current pharmacotherapy leave many chronic pain patients without adequate treatment due to negative physical and social side effects. Thus, there is a critical need to develop new pain management therapies with long-term effectiveness and minimal adverse effects. Using an adeno-associated virus (AAV) gene therapy to treat chronic pain has gained interest in the last twenty years because of several therapeutic advantages. AAV gene therapy allows for selective gene expression directly to sites of interest for chronic pain treatment with the potential for sustained expression following a single injection. In an effort to treat chronic pain using an AAV gene therapy, we have created a viral vector using recombinant adeno-associated virus which encodes the biosynthetic enzyme, human arginine decarboxylase (hADC). Arginine decarboxylase (ADC) is an endogenous enzyme that catalyzes the metabolism of L-arginine into agmatine. Agmatine is an endogenous small molecule that acts as a neurotransmitter and has been previously shown to modulate neuroplastic events by antagonizing the GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor. When agmatine is delivered centrally, a reduction in pain behavior following nerve injury is observed. The central hypothesis of this body of work is that overexpression of arginine decarboxylase using an adeno-associated viral vector construct results in long-term reduction of neuropathic pain due to the production of agmatine and subsequent antagonism of the GluN2B NMDA receptor by agmatine. The experiments presented in this dissertation build upon our agmatine-based pharmacotherapy for the treatment of chronic neuropathic pain. Specifically, this work furthers our AAV-based gene therapy by understanding intrathecal AAV kinetics, evaluating how therapeutic efficacy changes with age and with the use of cell-specific promoters, and interpreting associated behavioral implications.

Opioid-based pharmacotherapy remains the most commonly prescribed treatment for patients suffering from neuropathic pain conditions. Although opioids are effective for treating neuropathic pain, when used chronically the development of adverse side effects, such as opioid analgesic tolerance, can develop. Previous studies have shown that exogenously delivered agmatine, (decarboxylated L-arginine) can prevent the development of opioid analgesic tolerance, dependence, and self-administration. This study investigated the impact of intrathecal adeno-associated virus serotype 5-human arginine decarboxylase (AAV5-hADC) in models of opioid analgesic tolerance. Pharmacological dose-response curves were collected from two cohorts using two different models of opioid analgesic tolerance. Tissues from the central nervous system of the subjects were further analyzed for hADC gene expression and for spinal agmatine levels. Investigations of the choroid plexus as a target for intrathecal gene therapeutics were also conducted. Region-directed genetic modulation of the agmatine metabolic pathway within the central nervous system could be a highly innovative approach for the control of opioid tolerance and other neuroplasticity disorders.

Arginine decarboxylase is a key enzyme involved in the polyamine pathway of organisms. Pyruvoyl-dependent arginine decarboxylases are expressed in the form of proenzymes that self-cleave to form N-terminal [beta] and C-terminal [alpha] subunits generating an active pyruvoyl group at the [alpha] terminus. We have identified an archaeal homolog of a pyruvoyl-dependent arginine decarboxylase in Chlamydophila pneumoniae that could play a role in the persistence of the organism in the host. The recombinant enzyme showed highest activity at pH 3.4, which is the lowest optimum pH ever reported for a pyruvoyl dependent arginine decarboxylase. The proton-consuming decarboxylation raises intracellular pH, and thereby plays a role in acid-resistance. It could inhibit the pro-inflammatory nitric oxide synthase resulting in asymptomatic infection. A variant protein Thr⁵²Ser at the predicted cleavage site showed less pro-enzyme cleavage and activity compared to the wild-type. The homologs of arginine decarboxylase and flanking arginine-agmatine antiporter were also found in different biovariants of Chlamydia trachomatis. In the invasive L2 strain of C. trachomatis, the presence of a nonsense codon in the gene encoding arginine decarboxylase enzyme prevented the expression of an active enzyme. The variant protein with tryptophan replacing nonsense codon restored arginine decarboxylase activity. The non-invasive D strain of C. trachomatis had an intact arginine decarboxylase gene, but it was recombinantly expressed as a proenzyme that was uncleaved. The arginine-agmatine antiporters from both the strains were active and transported tritiated arginine into their cells. The polyamine pathway of the crenarchaeon Sulfolobus solfataricus uses arginine to make putrescine, but the organism lacks homologs of arginine decarboxylase. However, it has two paralogs of pyruvoyl dependent S-adenosylmethionine decarboxylase − SSO0536 and SSO0585. These enzymes were recombinantly expressed as pro-enzymes that self-cleaved into [beta] and [alpha] subunits. Even with a 47% amino acid sequence identity, the SSO0536 protein exhibited significant arginine decarboxylase activity whereas SSO0585 protein had significant S-adenosylmethionine decarboxylase activity. This is the first report of an S-adenosylmethionine decarboxylase enzyme showing alternative decarboxylase activity. The chimeric protein with the [alpha]-subunit of SSO0585 and [beta]-subunit of SSO0536 had arginine decarboxylase activity, suggesting that the residues responsible for substrate recognition are located in the amino terminus.