Nir Shapir

Preclinical and preliminary clinical evaluation of genetically transduced dermal tissue implants for the sustained secretion of erythropoietin and interferon α

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 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 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 five 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.

TARGT Gene Therapy Platform for Correction of Anemia in End-Stage Renal Disease

A new protein-delivery platform can provide sustained secretion of recombinant human erythropoietin with the use of small, transduced autologous dermal implants. Study patients with end-stage renal disease were found to have maintenance of stable hemoglobin levels.

Successful intracranial delivery of trastuzumab by gene-therapy for treatment of HER2-positive breast cancer brain metastases

Background: Trastuzumab is a monoclonal antibody which demonstrates efficacy for HER2 positive breast cancer patients. Recently, an increased incidence of brain metastasis in trastuzumab‐treated patients has been reported. The reason for this may be the effectiveness of systemic trastuzumab allowing patients to survive longer thus providing time for brain metastases to develop, along with the lack of penetration of systemic therapies through the blood brain barrier. In recent years, several administration routes to the brain have been evaluated. Albeit advances in the field, there is still a need for improved delivery of therapeutic antibodies to the brain. To address this challenge, we have developed two gene therapy‐based methods enabling continuous secretion of active trastuzumab in the brain. Methods: We have developed two gene therapy approaches for the delivery of the therapeutic anti‐HER2 monoclonal antibody, trastuzumab, to the brain. We utilized the helper dependent adenovirus vector, containing trastuzumab light and heavy chains coding sequences (HDAd‐trastuzumab). In the first approach, we used the Transduced Autologous Restorative Gene Therapy (TARGT) platform, in which dermal fibroblasts of human and mouse origin, are ex‐vivo transduced with HDAd‐trastuzumab vector, rendering continuous secretion of active trastuzumab from the cells locally. These genetically engineered cells were subsequently implanted intracranially to mice, contralateral to HER2 positive breast carcinoma cells inoculation site, enabling continuous secretion of trastuzumab in the brain. In the second approach, we used the same HDAd‐trastuzumab viral vector, directly injected intracranially, contralateral to the HER2 positive breast carcinoma cells inoculation site. Both methods enabled therapeutic concentrations of local in‐vivo production of active trastuzumab in a mouse model of brain metastatic breast cancer. Results: Trastuzumab secreted from the TARGT platform demonstrated in‐vitro affinity and immune recruitment activity (ADCC) similar to recombinant trastuzumab (Herceptin, Genentech). When implanted in the brain of HER2 positive tumor‐bearing mice, both the TARGT platform of dermal fibroblasts engineered to secrete trastuzumab and direct injection of HDAd‐trastuzumab demonstrated remarkable intracranial tumor growth inhibitory effect. Conclusions: This work presents two gene therapy approaches for the administration of therapeutic antibodies to the brain. The TARGT platform of dermal fibroblasts engineered to secrete active trastuzumab, and the direct injection of HDAd‐trastuzumab viral vector, both rendered continuous in‐vivo secretion of active trastuzumab in the brain and demonstrated high efficacy. These two approaches present a proof of concept for promising gene therapy based administration methods for intracranial tumors as well as other brain diseases.

Sustained secretion of anti-tumor necrosis factor α monoclonal antibody from ex vivo genetically engineered dermal tissue demonstrates therapeutic activity in mouse model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a symmetric inflammatory polyarthritis associated with high concentrations of pro‐inflammatory, cytokines including tumor necrosis factor (TNF)‐α. Adalimumab is a monoclonal antibody (mAb) that binds TNF‐α, and is widely used to treat RA. Despite its proven clinical efficacy, adalimumab and other therapeutic mAbs have disadvantages, including the requirement for repeated bolus injections and the appearance of treatment limiting anti‐drug antibodies. To address these issues, we have developed an innovative ex vivo gene therapy approach, termed transduced autologous restorative gene therapy (TARGT), to produce and secrete adalimumab for the treatment of RA.

598. TARGTCNS for the Treatment of Central Nervous System Disorders

Diseases and disorders of the central nervous system (CNS) represent a large field of unmet need that significantly impacts the lifespan and quality of life of many patients. The ability to deliver therapeutic proteins from the systemic circulation to the CNS is hampered by the blood-brain barrier (BBB). A number of strategies have been developed to allow therapies to cross the BBB, but to date with limited success. We have developed a novel localized approach to deliver protein and peptide therapeutics to the CNS based on our proprietary Transduced Autologous Restorative Gene Therapy system (TARGT™) platform. TARGT is based on autologous dermal micro-organs (MO) harvested and transduced ex-vivo to produce a therapeutic protein and then re-implanted in the patient. In this study we assessed whether TARGT can survive and deliver sustained localized protein post implantation into the CNS. In rats, MOs, 2×1mm each, were harvested from Lewis rat skin, characterized in-vitro, and then implanted in the Lewis rats’ cisterna magna. The procedure was well-tolerated and without observed behavioral change in all implanted rats. Histopathology of MOs excised several weeks post implantation, demonstrated viable and integrated MOs without signs of implant rejection. In the second phase, rat MOs were processed ex-vivo into TARGT secreting erythropoietin (TARGTEPO) by transduction with Helper Dependent Adenoviral vector encoding human erythropoietin (HDAd-HuEPO). Rat TARGTs secreting human EPO were then implanted into Lewis rats’ cisterna magna. Post implantation, cerebrospinal fluid (CSF) and serum were collected and EPO levels were measured by HuEPO ELISA. Results obtained suggest that implantation was well tolerated with no observed signs of rejection. Measurable human EPO levels were detected in rat CSF for the duration of the experiment. Detectable but low levels of Hu-EPO were also measured in the rat serum, as expected, since CSF drains into the peripheral blood. In pigs we have tested the viability and monoclonal antibody secretion from TARGTs into the CNS. Humira, an established antibody drug, was selected as a proof of concept. Pig MOs were harvested and transduced with the HDAd vector carrying the Humira sequence. Two TARGTHumira were implanted in the pig subdural space at the parietal convexity region and the pig was followed for one week post implantation. Results obtained post implantation suggests no observed pigs behavioral change. Humira was measured in CSF samples taken from the implantation area and the lumbar space. One week post implantation, TARGTs were excised out of the pig brain when they are viable and no signs of inflammation or damage to the brain tissue were observed. These results demonstrate that TARGT is a promising novel therapeutic platform with the potential for treatment of CNS disorders. We are now actively studying TARGTCNS treatment of lysosomal storage diseases and brain malignancies.

29. Clinical Trial Showing EPO-Independence for 7 Months by Prolonged Secretion of Autologous EPO by TARGT™

Recombinant human EPO (rHuEPO) along with iron supplementation corrects anemia in most patients with End StageRenal Disease (ESRD) but is associated with supra-physiological peak serum concentration (Cmax) of EPO and may cause thromboembolic complications.The Transduced Autologous Restorative Gene Therapy system (TARGT™) is an ex-vivo gene therapy that provides autologous, continuous proteins or peptide therapy in the physiological range. The system consists of several 2 × 30 mm pieces of dermal tissue (Micro-Organ, MO), extracted under local anesthetic in which its fibroblasts cells are transduced with a Helper-Dependent Adenoviral Vector (HDAd) containing the EPO gene expression cassette. After culture, and measurement of EPO production, one or more transduced MOs (TARGTs) are re-implanted as required for delivering the target dose. Patients are treated with local steroid injection to stabilize secretion. The system allows dose flexibility and the TARGTs may be removed or added according to the in-vivo secretion levels.We present here initial results from an-ongoing open label ascending dose clinical study of TARGTEPO in patients with anemia due to ESRD. We have completed the enrollment of patients in the first out of 3 cohorts (the low dose) with 6 EPO-dependent patients treated with a total of up to 3 TARGTepo units each, secreting a total of 25 IU/Kg/day of autologous EPO. All patients continued their previous regimen of intravenous supplemental iron.Patients follow-up post implantation is still ongoing with one patient being followed with stable EPO secretion and resulting stable Hb for over 7 months. Results obtained suggest stabilization of serum EPO levels at the physiological range of £20 mIU/ml and the resulting Hb levels between 9-12 g/dL without rHuEPO or transfusion while TARGTs are still functioning. Comparative analysis of serum EPO levels revealed significantly lower Cmax with TARGTepo compared to rHuEPO. Also, comparison of extrapolated Area Under the Curve (AUC) of rhEPO vs. actual TARGTepo AUC, revealed that TARGTepo maintained Hb within the desired range in patients with an order of magnitude smaller exposure to EPO compared to rHuEPO. This observation may have significant clinical benefits. No treatment related serious adverse events have been reported. TARGTEPO is a promising novel therapy for anemia and potentially for other protein deficient diseases.

180. TARGT™ Shows Sustained Secretion of the Therapeutic Peptide GLP-2 Which Retains Functional Activity in SCID Mice and Rats

Glucagon-like peptide-2 (GLP-2) is a naturally occurring 33 amino acid peptide, with t1/2 of 6-7 min. This peptide is produced by L cells of small intestine and has several important functions, such as increasing intestinal absorption, stimulating intestinal growth, reducing bone breakdown and impacting gastric and intestinal motility. It is well known that individuals with short bowel syndrome benefit from exogenous GLP-2 to improve intestinal absorption.Native peptides have been difficult to develop into drugs because of their short half-life, poor metabolic stability and rapid clearance. Manufactured peptides frequently require structural modification and, long-acting formulations. Moreover, the treatment with such peptides frequently uses high doses, which can lead to off-target effects such as nausea and vomiting in some patients. As an alternative, we developed a novel approach that allows our TARGT™ (Transduced Autologous Restorative Gene Therapy) technology to produce and secrete peptides such as GLP-2.The TARGT™ system is an ex-vivo gene therapy, which we have now demonstrated is capable of providing autologous, continuous peptide therapies, at physiological ranges. Our group previously demonstrated such findings for certain proteins. In one set of experiments described herein, the TARGT™ system consists of several 2 × 30 mm biopsies of dermal tissue (Micro-Organ, MO), extracted under local anesthetic followed by fibroblast transduction with a Helper-Dependent Adenoviral Vector (HDAd) containing a novel GLP-2 gene expression cassette. After culture, and measurement of peptide production, one or more transduced MOs (TARGTs™) are re-implanted into the patient as required to achieve the desired dose. The system allows dose flexibility and the TARGTs™ may be added or removed according to in-vivo secretion levels.Pre-clinical studies, using this novel technology, showed GLP-2 TARGT (TARGTGLP-2) in-vitro secretion levels of tens of micrograms per day. Preliminary data from in-vivo SCID mice experiments confirms that TARGT-secreted human GLP-2 analog reaches the mouse blood stream and exhibits a sustained secretion profile similar to tho se observed and reported with other proteins using the TARGT™ technology (e.g. EPO, IFNα). In addition, in-vivo SCID mice and rat experiments suggest that TARGT™ secreted GLP-2 analog is active, and increases intestinal villous height and intestinal crypt cell proliferation rate.Data obtained so far suggests that TARGTGLP-2 may provide a favorable PK profile and physiologic levels of continuous GLP-2 for extended periods of time, avoiding supra-physiological peak serum concentration and achieving continuous coverage with less exposure compared to exogenous GLP-2 injections. The current results suggest that the TARGT™ platform is a promising novel therapy for short bowel syndrome and potentially other diseases of endogenous protein/peptide deficiency.