Seeking a Cure for the Royal Pain

Recurrent debilitating episodes of acute abdominal pain, peripheral nerve paresthesia, and psychiatric symptoms including acute depression or psychosis are the hallmarks of the so-called hepatic porphyrias, a series of rare inherited disorders affecting the heme biosynthesis pathway. Acute intermittent porphyria (AIP) is, among both lay and medically trained individuals, likely the most well known, if not actually the most common, form of hepatic porphyria. King George III of England, probably the most famous person thought to have had porphyria, suffered during his reign from at least five episodes, each lasting weeks to months, of mental confusion, excitability, and insomnia in association with severe abdominal pain, diffuse muscle cramping and pain, burning sensations in his hands and feet, and discoloration of his urine.

Professors Macalpine and Hunter, in an extensive review of the available documentation concerning the health of “Mad King George,” suggested the diagnosis of AIP (Macalpine and Hunter, 1966), although reports of skin photosensitivity and “great weals on his arms” make another form of hepatic porphyria, namely variegate porphyria, more likely (Macalpine et al., 1968). Although the theory is not universally accepted (Hift et al., 2012) and lacks direct proof, porphyria-like symptoms in many descendants of George III have led to the hypothesis that several members of the British and former German royal families had inherited porphyria from the “Mad King” (Rohl et al., 1999). In this issue of Human Gene Therapy, Paneda and colleagues (2013) report progress in the development of recombinant adeno-associated virus (rAAV)-mediated liver-directed gene therapy for the treatment of AIP and announce plans for a Phase I clinical trial of this novel therapeutic approach. Perhaps a cure for the royal pain is in the offing.

AIP is caused by partial deficiency of porphobilinogen deaminase (PBGD) (also known as hydroxymethylbilane synthase, or HMBS), an intermediate step in the synthesis of heme, associated with dominantly inherited mutations in the PBGD gene. Although small amounts of heme are likely synthesized in most tissues, the activities of the heme biosynthetic enzymes including PBGD and the rate of heme production are greatest in erythroid bone marrow and liver. Heme of course is required for the production of oxygen-carrying hemoglobin in erythrocytes and myoglobin in muscle but is also a required cofactor for several enzymes with more ubiquitous expression including respiratory chain cytochromes and enzymes of the P450 detoxification system. The initial step in heme synthesis, namely the condensation of glycine with succinyl-CoA catalyzed by 5-aminolevulinate synthase (ALAS), is the rate-limiting step in the pathway. Induction of the P450 system leads to increased liver ALAS activity and consequently increased heme synthesis. Heme itself represses ALAS activity through regulatory action on both ALAS gene transcription and mRNA translation.

In the hepatic porphyrias, heme biosynthesis is likely sufficient to maintain normal physiologic function under basal conditions but under conditions of increased demand for heme, such as infection or administration of a drug metabolized in the P450 system, haploinsufficiency of a step in heme biosynthesis impairs the required response to the stressor. When ALAS activity is fully induced, the reduced activity of PBGD in patients with AIP becomes a serious bottleneck. The primary approach to contemporary therapy of AIP relies upon avoidance of known triggers including a large list of pharmaceuticals that induce P450 expression, yet debilitating porphyric crises, some causing permanent neurologic damage, recur.

The symptoms of AIP are virtually all the result of neurologic dysfunction, but the precise pathophysiologic mechanism underlying this dysfunction has not been fully elucidated. Local heme deficiency in affected neurons could play a role, but the preponderant evidence points to neurotoxic effects of the heme precursors, 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), produced in non-nervous tissues. Both ALA and PBG accumulate behind the bottleneck at PBGD during acute porphyric crises. In contemporary therapy of a porphyric crisis, intravenous infusion of heme (as hematin or heme arginate) suppresses liver ALAS activity, reduces ALA and PBG production, and gradually alleviates the neurologic symptoms associated with an AIP crisis. This outcome provides the rationale for liver gene therapy as a possible treatment for AIP: restoration of liver PBGD activity following liver-directed gene therapy would prevent accumulation of ALA and PBG during the induction of heme synthesis and consequently prevent the development of neurologic symptoms associated with ALA and PBG toxicity.

The success of orthotopic liver transplantation as therapy for AIP is further proof that liver-directed therapy can alleviate the neurologic phenotype (Soonawalla et al., 2004). Interestingly, liver PBGD expression from plasmid vector delivered by hydrodynamic tail vein injection to a murine AIP model corrected the metabolic phenotype while transplantation of wild-type bone marrow to these animals did not lead to metabolic correction (Unzu et al., 2010). Successful correction of liver PBGD activity, reduction of heme precursor concentrations, and amelioration of AIP-associated neurotoxicity following treatment of AIP mice with either serotype 8 or serotype 5 rAAV vectors has subsequently been reported by two different laboratories (Yasuda et al., 2010; Unzu et al., 2011).

In their latest report, Paneda and coworkers describe the results of administering by intravenous infusion an rAAV serotype 5 vector expressing PBGD from a human codon-optimized cDNA driven by a liver-specific promoter to cynomologous macaques. In animals receiving the highest dose of 5×10¹³ vector genomes (vg)/kg body weight, diffuse transduction of the liver was detected 30 days after vector infusion and human PBGD activity expressed from the transgene measured up to 50% of the native activity in macaque liver. No serious adverse events attributable to vector administration were reported. As expected, all animals developed circulating antibody against the AAV5 capsid but no anti-vector or anti-human PBGD T cell responses were detected. Comprehensive evaluation of vector integration into the host genome by linear amplification-mediated polymerase chain reaction (LAM-PCR) analysis revealed a very low frequency of integration without predilection to any specific integration hotspots.

Given these successful results, a Phase I human clinical trial of this rAAV5 vector is planned. The goal of course is to restore liver PBGD expression to near normal in individuals with AIP and thereby prevent the accumulation of neurotoxic precursors during periods of increased demand upon heme biosynthesis. The precise therapeutic threshold for successful therapy, that is, the degree of liver transduction and the level of liver PBGD expression that must be achieved to fully correct the metabolic defect, is yet unknown. Will restoration of liver PBGD activity to only 75 or 80% of normal be sufficient to prevent porphyric crises? Will all manifestations of the disorder be ameliorated by liver-targeted therapy alone? A careful clinical trial should begin to answer these questions. If successful for AIP, liver gene therapy would likely also be successful as a treatment for the other hepatic porphyrias. If so, then future descendants of King George III may benefit from a permanent cure for the “Royal Malady.”