CARATTERIZZAZIONE DEL DNA INCAPSIDATO IN VIRUS ADENO ASSOCIATI RICOMBINANTI PRODOTTI IN S.CEREVISIAE.

I virus ricombinanti sono molto utilizzati per veicolare DNA, a scopo terapeutico, all’interno di una cellula. Un virus che trova largo impiego in questo campo è il virus adeno-associato (AAV). AAV è un virus non patogeno e difettivo, della famiglia dei Parvovirus, che necessita di una co-infezione da parte di un virus helper (solitamente adenovirus) per stabilire un’infezione nell’ospite e replicarsi attivamente. In assenza di virus helper, AAV si integra in modo sito-specifico nel genoma dell’ospite, stabilendo un’infezione latente. Il suo genoma a singolo filamento di 4,7kb è composto da due ORFs, Rep e Cap, che codificano rispettivamente per le proteine della replicazione e del capside. Le ORFs sono fiancheggiate da due sequenze terminali, invertite e ripetute (ITR), essenziali sia per la replicazione che per l’incapsidazione del virus. Uno dei problemi legati all’uso di AAV ricombinante (rAAV) nella terapia genica è rappresentato dalla difficoltà di produzione. Per mettere a punto una nuova strategia di produzione di rAAV nel lievito S.cerevisiae è stato dimostrato che, in questo organismo, il single-strand DNA (ssDNA) di AAV è prodotto da un plasmide contenente le ITR e che l’espressione delle proteine del capside porta alla formazione di “virus like particles” (VLP). é stato inoltre osservato che del DNA viene incapsidato nelle VLPs. Il lavoro della mia tesi si propone di migliorare il metodo di purificazione delle particelle ricombinanti prodotte in lievito e di caratterizzare il DNA contenuto nelle VLPs. Dal momento che i vettori ricombinanti di AAV (rAAV) che vogliamo produrre in lievito devono essere utilizzati per veicolare DNA nelle cellule umane, abbiamo costruito un vettore in cui tra le ITRs è presente non solo il marcatore URA3 di lievito ma anche il gene che codifica per la “Green fluorescent protein” (GFP) sotto il controllo del promotore CMV creando così il plasmide pAAVGFPURA. Abbiamo trasformato RSY12 contenente il gene Rep integrato con pAAVGFPURA, pESCVP2,3VP1KM ed il plasmide per l’espressione di E4orf6 ed E1b55k. Le VLPs prodotte in questo ceppo, sono state estratte e sottoposte a purificazione con gradiente di iodixanolo, che consente di purificare rAAV in maniera più rapida rispetto al gradiente di CsCl utilizzato in precedenza. La purificazione eseguita è risultata essere ancora imperfetta dal momento che nella frazione analizzata non erano presenti le VLP. Era stato osservato precedentemente in laboratorio che il DNA viene incapsidato nelle VLP ma non era stato caratterizzato. Pertanto in questo lavoro abbiamo caratterizzato il DNA incapsidato e cercato di capire quanto ssDNA prodotto nella cellula venisse effettivamente incluso nelle VLPs. A tale scopo, siamo partiti da un ceppo già creato in laboratorio, contenente una delezione completa del gene URA3 ed il gene Rep integrato nel genoma, trasformato con il plasmide pAAVpokURA2μ, contenente la sequenza spaziatrice (pok) e URA3 (gene marcatore) fiancheggiate dalle ITR. Abbiamo poi indotto la produzione di proteine virali VP1, 2 e 3, trasformando il ceppo con un secondo plasmide, pESCVP2,3VP1KM, che consente l’espressione di tali proteine sotto il controllo di due promotori inducibile da galattosio (pGAL1 e pGAL10). La contemporanea presenza nella cellula di ssDNA e proteine del capside determina l’incapsidazione di DNA nelle VLPs. Per quantificare il DNA incapsidato abbiamo effettuato una real time PCR (RT-qPCR), ma i risultati ottenuti hanno indicato che il metodo non consente di misurare diverse quantità di ssDNA in presenza o assenza di capsidi

Fibroblasts Impair Migration and Antitumor Activity of NK-92 Lymphocytes in a Melanoma-on-Chip Model

Adoptive cell therapy in solid tumors, such as melanoma, is impaired, but little is known about the role that the fibroblasts present in the tumor microenvironment could exert. However, the mechanism at play is not well understood, partly due to the lack of relevant pre-clinical models. Three-dimensional culture and microfluidic chips are used to recapitulate the dynamic interactions among different types of cells in the tumor microenvironment in controlled and physiological settings. In this brief report, we propose a reductionist melanoma-on-a-chip model for evaluating the essential role of fibroblasts in the antitumor activity of lymphocytes. To this end, 3D melanoma spheroids were monocultured and co-cultured with human dermal fibroblasts and the NK-92 cell migration towards the tumor compartment was tested in a commercially available microfluidic device. Utilizing confocal microscopy, we observed the different recruitment of NK-92 cells in the presence and absence of fibroblasts. Our results show that fibroblasts’ presence inhibits immune effector recruiting by exploiting a 3D pre-clinical tumor model

Recruitment, Infiltration, and Cytotoxicity of HLA-Independent Killer Lymphocytes in Three-Dimensional Melanoma Models

Cancer adoptive cell therapy (ACT) with HLA-independent tumor killer lymphocytes is a promising approach, with intrinsic features potentially addressing crucial tumor-escape mechanisms of checkpoint inhibitors. Cytokine-induced Killer (CIK) and Natural Killer (NK) lymphocytes share similar tumor-killing mechanisms, with preclinical evidence of intense activity against multiple solid tumors and currently testing in clinical studies. To improve the effective clinical translation of such ACT approaches, several fundamental questions still need to be addressed within appropriate preclinical contexts, capable of overcoming limitations imposed by most traditional two-dimensional assays. Here, we developed a novel experimental approach to explore, dissect, and visualize the interactions of CIK and NK lymphocytes with melanoma tumors in vitro in 3D. Primary melanoma cells were assembled into small tumors that were dispersed in a 3D matrix and challenged with patient-derived CIK or the NK-92 cell line. By means of imaging-based methods, we reported, visualized, and quantitatively measured the recruitment of CIK and NK on the 3D targets, their infiltration, and cytotoxic activity. Our results support the effective tumor recruitment and tumor infiltration by CIK and NK. Such features appeared dependent on the specific geometric aspects of the environment but can be explained in terms of directional migration toward the tumor, without invoking major feedback components. Overall, our 3D platform allows us to monitor the processes of tumor recruitment, infiltration, and killing by means of live measurements, revealing important kinetic aspects of ACT with CIK and NK against melanoma

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes

Purpose: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice.Experimental Design: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models.Results: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK.Conclusions: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma

Abstract Background Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40–60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. Methods In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. Results We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. Conclusions In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors