Renato Minopoli

Telethon Institute Of Genetics And Medicine

Author Statistics

Papers

Citation

H-Index

i-10 index

Research Trends

Author Order

Document Type

Co-Authors

Alberto Auricchio

Federico II University Hospital

Ivana Trapani

Telethon Institute Of Genetics And Medicine

Patrizia Tornabene

Cincinnati Children's Hospital Medical Center

Carolina Iodice

Telethon Institute Of Genetics And Medicine

Enrico Maria Surace

University of Naples Federico II

Settimio Rossi

University of Campania "Luigi Vanvitelli"

Francesca Simonelli

University of Campania "Luigi Vanvitelli"

Miriam Centrulo

Telethon Institute Of Genetics And Medicine

Andrea Maddalena

University of Bern

Elena Marrocco

Telethon Institute Of Genetics And Medicine

Cooperative Institutions

Telethon Institute Of Genetics And Medicine

University of Naples Federico II

Federico II University Hospital

University of Campania "Luigi Vanvitelli"

National Research Council

Radboud University Nijmegen

Radboud University Medical Center

Telethon Foundation

Sapienza University of Rome

Istituti di Ricovero e Cura a Carattere Scientifico

Author Statistics

Papers

Citation

H-Index

i-10 index

Research Field

Triple Vectors Expand AAV Transfer Capacity in the Retina

Molecular Therapy (2017)

Andrea Maddalena Patrizia Tornabene Paola Tiberi Renato Minopoli Anna Manfredi

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

Transduction (biophysics)

Gene transfer

Adeno-associated virus

10.1016/j.ymthe.2017.11.019

Cite

Citations (105)

Therapeutic homology-independent targeted integration in retina and liver

Nature Communications (2022)

Patrizia Tornabene Rita Ferla Manel Llado Miriam Centrulo Margherita Dell’Anno

Abstract Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.

10.1038/s41467-022-29550-8

Cite

Citations (28)

Triple AAV vectors to expand AAV cargo capacity in the retina

Investigative Ophthalmology & Visual Science (2015)

Andrea Maddalena Pasqualina Colella Ivana Trapani Renato Minopoli Carolina Iodice

Source

Cite

Citations (1)

Inclusion of a degron reduces levels of undesired inteins after AAV-mediated protein trans-splicing in the retina

Molecular Therapy — Methods & Clinical Development (2021)

Patrizia Tornabene Ivana Trapani Miriam Centrulo Elena Marrocco Renato Minopoli

Split intein-mediated protein trans-splicing expands AAV transfer capacity, thus overcoming the limited AAV cargo. However, non-mammalian inteins persist as trans-splicing by-products, and this could raise safety concerns for AAV intein clinical applications. In this study, we tested the ability of several degrons to selectively decrease levels of inteins after protein trans-splicing and found that a version of E. coli dihydrofolate reductase, which we have shortened to better fit into the AAV vector, is the most effective. We show that subretinal administration of AAV intein armed with this short degron is both safe and effective in a mouse model of Stargardt disease (STGD1), which is the most common form of inherited macular degeneration in humans. This supports the use of optimized AAV intein for gene therapy of both STGD1 and other conditions that require transfer of large genes.

Intein

Degron

Protein splicing

10.1016/j.omtm.2021.10.004

Cite

Citations (17)

187. TFEB-Mediated Clearance of the Lipofuscin Fluorophore A2E

Molecular Therapy (2016)

Ivana Trapani Elisabetta Toriello Renato Minopoli Alberto Auricchio

Abnormal accumulation of various by-products of the visual cycle, including the diretinoid-pyridinium-ethanolamine (A2E), in retinal pigment epithelium (RPE) is an hallmark of both Stargardt disease (STGD) and age-related macular degeneration (AMD). This is responsible for RPE and, consequently, photoreceptor (PR) cell death. A2E storage in RPE lysosomes has been shown to reduce both the capacity of RPE to degrade phagocytized PR outer segments and autophagosome biogenesis, trafficking and autophagic flux. The transcription factor EB (TFEB) is a master regulator of cellular clearance. Here we tested if TFEB overexpression induces A2E clearance from the RPE cells. We have generated a plasmid encoding for a double-serine mutant (S142A, S211A) form of TFEB, which is constitutively active. We have transfected this plasmid in the human RPE-derived ARPE19 cells to evaluate TFEB activation of its transcriptional targets and ability to clear A2E after loading. We found that TFEB overexpression in ARPE19 cells results in the induction of TFEB transcriptional targets involved in cellular clearance. Importantly, TFEB overexpression in ARPE19 cells was associated with reduction of intracellular A2E accumulation. Taken together these results suggest that TFEB overexpression is an effective strategy to promote A2E clearance in vitro. Further studies will clarify if this holds true in vivo. These results may have implications for the therapy of both STGD and the more common AMD.

TFEB

10.1016/s1525-0016(16)32996-3

Cite

Citations (0)

500. Triple AAV Vectors Expand AAV Cargo Capacity in the Retina

Molecular Therapy (2015)

Andrea Maddalena Pasqualina Colella Ivana Trapani Renato Minopoli Alberto Auricchio

We have recently shown that dual AAV vectors expand AAV cargo capacity in the retina up to 9kb thus allowing their application to the therapy of inherited blinding conditions like Stargardt disease or Usher syndrome type IB (USHIB). However, for several other inherited blinding conditions the size of the coding sequence to be transferred would not fit into dual AAV vectors and would require a triple AAV vector system. We have generated triple AAV vectors encoding a reporter EGFP-DsRed fusion protein under the transcriptional control of the ubiquitous CMV promoter. The expression cassette was split in three parts and packaged in three distinct AAV vectors. In order to favour the directional concatemerization upon co-infection, highly recombinogenic sequences were added to the constructs which would then be eliminated from the mature transcript by the inclusion of flanking splicing signals (Figure 1). Triple AAV 2/2 vectors were used to infect HEK293 cells in vitro and AAV 2/8 vectors in vivo by subretinal administration in C57Bl/6 mice. Direct fluorescence analysis showed EGFP and DsRed colocalization in HEK293 cells; full length protein expression was confirmed by Western Blot analysis of HEK293 cell lysates. Fundoscopy analysis of C57Bl/6 mice up to 2 months after subretinal delivery shows signal compatible with EGFP-DsRed expression. Western-blot analysis of retinal lysates supports the production of the full length protein. Our results suggest that AAV DNA cargo capacity can be increased up to 12kb in the retina by triple AAV vectors. This bodes well for gene therapy of retinal diseases which require a high DNA cargo capacity.

HEK 293 cells

Adeno-associated virus

Expression cassette

Colocalization

10.1016/s1525-0016(16)34109-0

Cite

Citations (1)

Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina

Science Translational Medicine (2019)

Patrizia Tornabene Ivana Trapani Renato Minopoli Miriam Centrulo Mariangela Lupo

Adeno-associated viral vectors reconstitute large proteins in the retina via intein-mediated protein trans-splicing.

Intein