Adoptive transfer of T cells with transgenic antigen-specific T cell receptors (TCRs) has the potential to therapeutic options of infectious diseases and cancer. However, generation of defined T cell products with reliable functionality still poses a major challenge. Conventionally, genetic engineering by viral transduction is used, which leads to un-targeted transgene integration and interference through the endogenous TCR. Furthermore, careful titration of viral transduction doses is necessary to limit potential off-target effects through multiple integrations. We recently reported that CRISPR/Cas9-mediated orthotopic TCR replacement (OTR) offers the possibility of controlled transgene integration and simultaneous removal of the endogenous TCR. By genetic engineering of many different antigen-specific TCRs, we now show that knock-out of the endogenous TCR and targeted insertion through OTR independently contribute to highly defined transgenic TCR expression. In comparison to low-dose virally transduced TCR transgenic T cells, enhanced and more defined TCR expression through OTR leads to higher functionality and to a less variable T cell responses. Therefore, OTR seems indeed advantageous for the generation of more predictable TCR-transgenic T cell products.

We have recently discovered a novel subset of CD4+ T cell that express the high affinity receptor for IgG (FcgRI) in both mouse and human and efficiently kill tumor cells coated with antibodies. While the factors that induce these cells are currently unknown, we recapitulate their killing capacity by transducing conventional CD8+ T cells with altered FcgRI along with its signaling chain. The unique design of this genetic construct enables integration of complex signals, which are impossible to transmit using conventional CAR designs, thus inducing more potent cytotoxic activity. Moreover, since targeting tumor cells is mediated by antibodies, this technology can be applied to treat a wide range of cancers as well as refractory cancers that lost their expressed antigen, by changing the tumor-binding antibody.

In this talk I will describe cutting-edge technologies for engineering functional cardiac tissues and whole hearts. I will focus on the design of new biomaterials mimicking the natural microenvironment, or releasing biofactors to promote stem cell recruitment and tissue protection. In addition, I will discuss the development of patient-specific materials and 3D-printing of personalized vascularized tissues and organs. Finally, I will show a new direction in tissue engineering, where micro and nanoelectronics are integrated within engineered tissues to form cyborg tissues.

High-grade serous carcinoma (HGSC) is a type of epithelial ovarian cancer (EOC). EOC is the fourth most common cause of cancer-related death in women in the developed world and the leading cause of death from gynecological malignancies.  EOC is often diagnosed at advanced-stage disease with an overall 5-year survival rate of <40%. While a significant progress has been made in surgical and chemotherapeutic treatments for EOC, the survival rates for this disease have only modestly improved. We constructed a ‘dual-Chimeric Antigen Receptor’ (dCAR) for ovarian cancer. The dCAR represent a new approach that addresses the major challenge of 'off-tumor on-target' toxicity in CAR therapy, namely, the risk of damage to healthy tissues of the patient which express the target antigen of the selected CAR. The underlying idea is that restricting the activity of CAR T cells against a combination of two antigens which are co-expressed by the tumor, but not by normal tissues, can prevent off target toxicity. We analyzed the potential efficacy and safety of the dual CAR in vitro. Additionally, we compared the administration of CAR T cells locally to intra venues injection and compared it to i.v injections. We show that intra- tumoral injection have significant superior effect in vivo. 

Modifications of RNA affect its function and stability. RNA editing is unique among these modifications because it not only alters the cellular fate of RNA molecules but also alters their sequence relative to the genome. The most common type of RNA editing is A-to-I editing by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes. Recent transcriptomic studies have identified a number of ‘recoding’ sites at which A-to-I editing results in non-synonymous substitutions in protein-coding sequences. However, systematic mapping of the editome across the animal kingdom has revealed that most A-to-I editing sites are located within mobile elements in non-coding parts of the genome. Editing of these non-coding sites is thought to have a critical role in protecting against activation of innate immunity by self-transcripts. Both recoding and non-coding events have implications for genome evolution and, when deregulated, may lead to disease. Finally, ADARs are now being adapted for RNA engineering purposes.

Alternative splicing (AS) involves the selective inclusion and exclusion of exons from a nascent pre-mRNA that results in various protein sequences. Activated T cells display dynamics in isoform ratios of immune modulatory receptors and of splicing factors. Splicing variants of immune receptors may act in opposite directions but splicing factors are the major category of proteins that undergoes splicing shifts. By the use of splice-switching antisense oligonucleotides or splice-disrupting gene edits, a new avenue of transcriptional manipulations opens up to produce T cells with improved tumoricidal capacity for successful implementation of adoptive cell therapy against cancer.

Reut Horev1,2, Nisim Perets1,2, Yona Gefen3, Daniel Offen1,2

1Sackler School of Medicine, 2Sagol School of Neuroscience, Tel Aviv University, 3Stem Cell Medicine Ltd, Israel

Autism Spectrum disorder (ASD) is a neurodevelopmental disorder, mainly characterized by impairment of social interaction, communication deficits, and restricted interests. Currently, there is no pharmacologic medication that can reverse ASD symptoms. Using Shank3 InsG3680 mouse model of autism, mutation that present in 1% of ASD, we show that intranasal administration of exosomes derived from adipose mesenchymal stem cells has a remarkable ability to improve autistic-like behaviors. The treatment shows significant improvement in social interaction, communication, and risk assessment that preserved six months after single intranasal administration. Thus, our results suggest that non-invasive exosomes treatment might be used for ameliorating autistic-like phenotypes in patients.

Authors:
Banin, Eyal1, Reimann, Christopher2, Barak, Adiel3; Boyer, David4; Ehrlich, Rita5; Jaouni, Tareq2; McDonald, Richard6; Telander, David G.7; Keane, Michael8; Ackert, Jessica8; Ferguson, Mark D.8; Shabat, Avi Ben-9; Monés, Jordi M.10; Velez, Joyce11; Hogge, Gary S.11; Reubinoff, Benjamin12

Institutions:

1. Center for Retinal and Macular Degenerations, Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. 2. Cincinnati Eye Institute & University of Cincinnati School of Medicine, Cincinnati, Ohio, USA. 3. Sourasky Medical Center, Tel Aviv, Israel. 4. Retina Vitreous Associates Medical Group, Los Angeles, CA, USA. 5. Rabin Medical Center, Petah Tikva, Israel. 6. West Coast Retina Group, San Francisco, CA, USA. 7. Retinal Consultants Medical Group, Sacramento, CA, USA. 8. Gyroscope Therapeutics, Ambler, PA, USA. 9. Lineage Cell Therapeutics, Inc. (Cell Cure Neurosciences, Ltd.,) Jerusalem, Israel. 10. Institut de la Màcula, Barcelona, Spain. 11. Lineage Cell Therapeutics, Inc., Carlsbad, CA, USA. 12. Center for Embryonic Stem Cells Goldyn Savad Institute of Gene Therapy and the Department of Gynecology and Obstetrics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

Transplantation of RPE cells may be of therapeutic benefit in AMD. We developed RPE cells from hESCs using cGMP directed differentiation. Safety and tolerability of these cells is being evaluated in a Phase I/IIa clinical study in patients with dry AMD and geographic atrophy (GA) (NCT02286089). We report accumulated safety and imaging data from 17 subjects. 
RPE cells in suspension (OpRegen; 50-200k) were subretinally transplanted under local anesthesia to the worse vision eye using either pars plana vitrectomy (PPV) and retinotomy or via an alternative surgical approach utilizing Gyroscope's Orbit Subretinal Delivery System. RPE cells ready for onsite thawing and immediate transplantation have also been evaluated. Short course perioperatively systemic immunosuppression is used. Systemic and ocular safety is closely observed, and retinal function and structure are monitored using various imaging modalities.
Treatment has been well tolerated and there have been no unexpected adverse events (AEs) or treatment-related systemic serious AEs.  Visual improvement has been noted in all patients of cohort 4. In several subjects, within the area of RPE cell transplant, improvements of the ellipsoid zone and RPE layers at the border of GA, as well as directional growth changes in the area of GA, has been seen. Persistent changes observed following treatment include, alterations in drusen appearance, subretinal pigmentation and hyper-reflective areas, suggestive of the presence of transplanted RPE cells. In conclusion, subretinal transplantation of hESC-derived RPE cells in patients with dry AMD and GA appears well tolerated. Imaging findings suggest presence of transplanted cells in the subretinal space. Encouraging structural and clinical changes observed in some patients will require additional follow-up

Despite the remarkable efficiency of immune checkpoint modulators against metastatic melanoma, there is a low percentage of responders and clinical trials report severe immune-mediated side effects and disease relapse. Recent evidences show that non-tumor cells within the tumor microenvironment (TME), including tumor vasculature and immune stromal cells, dictate the overall therapeutic efficacy. We synthesized off-the-shelf and cost-effective nano-sized polymeric platform that combines a cancer vaccine with the targeted inhibition of molecular and/or cellular immune suppressive players. These precision nano-sized medicines aim to re-educate and harness patient T-cell response against tumors, leading to an immunological memory able to control tumor relapse without any follow-up treatment. The design of these advanced immunotherapies is guided by the identification of lead immune suppressor factors and tumor specific antigens using novel 3D bio-printed tumor-immune spheroids developed in our lab. Our first nano-immunotherapy candidates sensitized melanoma mouse models to immune-checkpoint modulators, dramatically increasing disease-free survival rates.