We describe a novel, two-nanoparticle mRNA delivery system and show that it is highly effective as a means of intracellular enzyme replacement therapy (i-ERT) using a murine model of ornithine transcarbamylase deficiency (OTCD). Our Hybrid mRNA Technology delivery system (HMT) comprises an inert lipid nanoparticle that protects the mRNA from nucleases in the blood as it distributes to the liver and a polymer micelle that targets hepatocytes and triggers endosomal release of mRNA. This results in high-level synthesis of the desired protein specifically in the liver. HMT delivery of human OTC mRNA normalizes plasma ammonia and urinary orotic acid levels, and leads to a prolonged survival benefit in the murine OTCD model. HMT represents a unique, non-viral mRNA delivery method that allows multi-dose, systemic administration for treatment of single-gene inherited metabolic diseases.

Thyroxine (T4) undergoes dynamic daily cycles in the perciform fish the red drum, Sciaenops ocellatus, that are inversely timed to cycles of thyrotropin (TSH) subunit mRNA expression in the pituitary gland. We have proposed that these daily cycles are regulated by negative feedback of circulating T4 on expression of pituitary thyroid hormone deiodinase type 3 (Dio3), such that elevated circulating T4 results in diminished pituitary thyroid hormone catabolism and consequent increased negative feedback on expression of TSH subunits during the day. To determine whether thyroid hormones function to modulate expression of pituitary deiodinase enzymes we developed an immersion technique to administer physiological doses of T3 and T4 in vivo. Immersion in T4 or T3 significantly inhibited the mRNA expression of the TSH α and β subunits from 4 to 66 h of immersion. Pituitary Dio3 expression was significantly diminished by T3 and T4 at 22 h. These results indicate that both T4 and T3 are capable of negative feedback regulation of TSH subunit expression in red drum at physiological concentrations and on a time scale consistent with the T4 daily cycle. Furthermore, thyroid hormones negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative thyroxine feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in other species.

Messenger RNA (mRNA) is a promising alternative in both the viral and non-viral DNA-based gene delivery fields. Current viral vectors for gene therapy are associated with serious safety concerns and nonviral vectors are limited by low gene transfer efficiency. mRNA gene expression in the liver can be used for treatment of genetic diseases involving disorders of metabolism. The majority are due to defects of single genes that code for enzymes expressed solely or predominantly in the liver. SMARTT Polymer Technology® has been developed into a robust platform for RNA therapeutics. Optimization of the mRNA technology platform has led to stepwise improvements in mRNA delivery to the liver using GalNAc targeted polymers. We demonstrated a 5,000-fold improvement in activity over our first generation delivery system. Urea cycle disorders result from single gene mutations that lead to deficiency in one of the six enzymes in the urea cycle pathway. This deficiency can trigger elevated blood ammonia levels, also known as hyperammonemia, a life-threatening illness that leads to brain damage, coma or even death in humans. The deficient protein is intracellular and IV protein therapeutics are ineffective. Liver transplantation is the only cure for urea cycle disorders but is limited by the shortage of donors and complications associated with rejection and infection of the transplant. There is a dire need for new treatment options. Using the mRNA technology platform, we have demonstrated preclinical proof of concept in a urea cycle disorder mouse disease model. Treatment with therapeutic mRNA shows normalization of blood ammonia levels in hyperammonemic mice. Therapeutic mRNA expression is detected in the liver after a single mRNA dose with good duration of expression. The treatment was well tolerated, with no toxicities associated with both single and multiple dosing regimens. This mRNA technology platform provides a significant opportunity for the treatment of urea cycle disorders and other orphan liver diseases.

Insufficient drug uptake by solid tumors remains the major problem for systemic chemotherapy. Many studies have demonstrated anticancer drug effects to be dose-dependent, although dose-escalation studies have resulted in limited survival benefit with increased systemic toxicities. One solution to this has been the idea of loco-regional drug treatments, which offer dramatically higher drug concentrations in tumor tissues while minimizing systemic toxicity. Although loco-regional delivery has been most prominent in cancers of the liver, soft tissues and serosal peritoneal malignancies, survival benefits are very far from desirable. This review discusses the evolution of loco-regional treatments, the present approaches and offers rapidly reversible hydrophobization of drugs as the new future direction.

We have investigated a rapidly reversible hydrophobization of therapeutic agents for improving first-pass uptake in locoregional drug therapy. This approach involves the attachment of a hydrophobic moiety to the drug by highly labile chemical linkages that rapidly hydrolyze upon injection. Hydrophobization drastically enhances cell-membrane association of the prodrug and, consequently, drug uptake, while the rapid lability protects nontargeted tissues from exposure to the highly active agent. Using the membrane-impermeable DNA intercalator propidium iodide, and melphalan, we report results from in vitro cellular internalization and toxicity studies. Additionally, we report in vivo results after a single liver arterial bolus injection, demonstrating both tumor targeting and increased survival in a mouse tumor model.

A critical step for liver-directed gene therapy is the selective targeting of nucleic acids to hepatocytes. We have previously discovered that the proximal half of the T7 phage tail fiber protein (p17) targeted intact T7 phage and recombinant proteins to hepatocytes in vivo. In the present study, we have localized the targeting activities to a 33 amino acid sequence within the p17 coiled-coil rod domain. Given that the tail fiber domain from which the peptide was derived may form alpha and triple helical structures, biophysical studies (CD spectra and analytical ultracentrifugation) were conducted to determine the secondary and tertiary structures of the peptide. This peptide is able to target proteins, polymers, and siRNA and also particles such as DNA polyplexes and liposomes to hepatocytes. A variety of coupling strategies and chemistries were employed, thus demonstrating that this peptide is a versatile system for delivering cargo. The ability of this hepatocyte-targeting peptide to target DNA-containing particles suggests that it should be useful in the development of both nonviral and viral vectors. However, biological function of delivered cargo has not been demonstrated. This was primarily due to failure of delivered cargo to escape the endosomes. Further studies are in progress to provide functional activity of delivered nucleic acids by enabling their endosomal escape.