ß-thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (i.e. using hepcidin activators like Tmprss6-antisense oligonucleotides (ASO)) or increase erythropoiesis (by erythropoietin (EPO) administration or by modulating the ability of transferrin receptor 2 (Tfr2) to control red blood cell (RBC) synthesis). Targeting Tmprss6 mRNA by Tmprss6-ASO was proven to be effective in improving the IE and splenomegaly by inducing iron restriction. However we postulated that combinatorial strategies might be superior to single therapies. Here we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by ß-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single allele deletion alone, respectively, exacerbated or did not improve the splenomegaly in ß-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve the splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, combination of Tmprss6-ASO+EPO or Tmprss6-ASO+Tfr2 single allele deletion showed significantly higher hemoglobin levels as well as reduction of splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating the IE and anemia in ß-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

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Protein drugs are currently delivered by bolus injection and although treatment frequently is successful, these methods also have major drawbacks, which call for the development of alternative technologies allowing prolonged delivery of these drugs. We developed a new ex vivo gene therapy platform called Transduced Autologous Restorative Gene Therapy (TARGT) for sustained long term production and secretion of autologous therapeutic proteins. A biopsy of dermal tissue taken from the patient is transduced ex vivo with a viral vector encoding the required gene under a constitutive promoter. Following measurement of protein secretion ex vivo, the transduced dermal tissue is implanted back into the patient, where it secretes the therapeutic protein into the circulation for several months or longer. A major hurdle to this approach is potential immunogenicity of the transduced tissue following implantation. In this paper we describe the preclinical and early clinical development of this technology, which allowed for overcoming these hurdles. To that end, we have used the helper dependent (HD) adenoviral vector with newly designed expression cassette containing genetic elements to optimize transgene expression. Moreover, we have developed procedures for TARGT tissue implantation, with measures to improve engraftment and reduce inflammation and rejection. Implantation of human TARGT to severe combined immune deficient (SCID) mice indicated long-term production of active proteins in the blood. Preliminary results of a clinical trial from two anemic end-stage renal disease patients, implanted with TARGTs expressing the human erythropoietin (EPO) gene, demonstrated prolonged secretion with physiologic blood level of the hormone and hemoglobin maintenance in the desired range, for a period of at least 5 months without exogenous EPO administration. We believe that the TARGT technology has the potential to become a platform for the sustained delivery of therapeutic proteins in various clinical indications.

KRAS mutations, which are the main cause of the pathogenesis of lethal pancreatic adenocarcinomas, impair the functioning of the GTPase subunit, thus rendering it constitutively active and signaling intracellular pathways that end with cell transformation. In the present study, the AsPC-1 cell line, which has a G12D-mutated KRAS gene sequence, was utilized as a cellular model to test peptide nucleic acid-based antisense technology. The use of peptide nucleic acids (PNAs) that are built to exhibit improved hybridization specificity and have an affinity for complementary RNA and DNA sequences, as well as a simple chemical structure and high biological stability that affords resistance to nucleases and proteases, enabled targeting of the KRAS-mutated gene to inhibit its expression at the translation level. Because PNA-based antisense molecules should be capable of binding to KRAS mRNA sequences, PNAs were utilized to target the mRNA of the mutated KRAS gene, a strategy that could lead to the development of a novel drug for pancreatic cancer. Moreover, it was demonstrated that introducing new PNA to cells inhibited the growth of cancer cells and induced apoptotic death and, notably, that it can inhibit G12D-mutated KRAS gene expression, as demonstrated by RT-PCR and western blotting. Altogether, these data strongly suggest that the use of PNA-based antisense agents is an attractive therapeutic approach to treating KRAS-driven cancers and may lead to the development of novel drugs that target the expression of other mutated genes.