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

2019 ◽  
Vol 11 (492) ◽  
pp. eaav4523 ◽  
Author(s):  
Patrizia Tornabene ◽  
Ivana Trapani ◽  
Renato Minopoli ◽  
Miriam Centrulo ◽  
Mariangela Lupo ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Virus Transfer ◽  
Split Intein ◽  
Trans Splicing ◽  
Effective Delivery ◽  
Cargo Capacity ◽  
Large Genes ◽  
Protein Reconstitution ◽  
Retinal Gene Therapy

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein–mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

scholarly journals Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

Gene Therapy ◽  
2013 ◽  
Vol 20 (8) ◽  
pp. 824-833 ◽  
Author(s):  
V S Lopes ◽  
S E Boye ◽  
C M Louie ◽  
S Boye ◽  
F Dyka ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Retinal Gene Therapy

Efficient Trans-Splicing in the Retina Expands the Utility of Adeno-Associated Virus as a Vector for Gene Therapy

2003 ◽  
Vol 14 (1) ◽  
pp. 37-44 ◽  
Author(s):  
S.J. Reich ◽  
A. Auricchio ◽  
M. Hildinger ◽  
E. Glover ◽  
A.M. Maguire ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adeno Associated Virus ◽  
Trans Splicing

scholarly journals Efficacy and Safety of Retinal Gene Therapy Using Adeno-Associated Virus Vector for Patients With Choroideremia

2019 ◽  
Vol 137 (11) ◽  
pp. 1247 ◽  
Author(s):  
M. Dominik Fischer ◽  
G. Alex Ochakovski ◽  
Benjamin Beier ◽  
Immanuel P. Seitz ◽  
Yousof Vaheb ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Virus Vector ◽  
Efficacy And Safety ◽  
Adeno Associated Virus ◽  
Retinal Gene Therapy

scholarly journals Semisynthesis of functional transmembrane proteins in GUVs

2021 ◽  
Author(s):  
K. A. Podolsky ◽  
T. Masubuchi ◽  
G. T. Debelouchina ◽  
E. Hui ◽  
N. K. Devaraj
Keyword(s):  
Cell Communication ◽  
Membrane Interface ◽  
Synthetic Membranes ◽  
Native Peptide ◽  
Split Intein ◽  
Trans Splicing ◽  
Pharmacology Biochemistry ◽  
Membrane Protein Reconstitution ◽  
Cell Death Protein ◽  
Protein Reconstitution

AbstractCellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

scholarly journals Relationship Between Neutralizing Antibodies Against Adeno-Associated Virus in the Vitreous and Serum: Effects on Retinal Gene Therapy

2019 ◽  
Vol 8 (2) ◽  
pp. 14 ◽  
Author(s):  
Suhwan Lee ◽  
Im Kyeung Kang ◽  
Ji Hyun Kim ◽  
Bok Kyoung Jung ◽  
Keerang Park ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Neutralizing Antibodies ◽  
Adeno Associated Virus ◽  
Retinal Gene Therapy

scholarly journals Battling Neurodegenerative Diseases with Adeno-Associated Virus-Based Approaches

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 460
Author(s):  
Olja Mijanović ◽  
Ana Branković ◽  
Anton V. Borovjagin ◽  
Denis V. Butnaru ◽  
Evgeny A. Bezrukov ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Neurodegenerative Diseases ◽  
Experimental Medicine ◽  
Target Cells ◽  
Adeno Associated Virus ◽  
Vector Systems ◽  
Effective Delivery ◽  
Broad Variety ◽  
In Vitro Data

Neurodegenerative diseases (NDDs) are most commonly found in adults and remain essentially incurable. Gene therapy using AAV vectors is a rapidly-growing field of experimental medicine that holds promise for the treatment of NDDs. To date, effective delivery of a therapeutic gene into target cells via AAV has been a major obstacle in the field. Ideally, transgenes should be delivered into the target cells specifically and efficiently, while promiscuous or off-target gene delivery should be minimized to avoid toxicity. In the pursuit of an ideal vehicle for NDD gene therapy, a broad variety of vector systems have been explored. Here we specifically outline the advantages of adeno-associated virus (AAV)-based vector systems for NDD therapy application. In contrast to many reviews on NDDs that can be found in the literature, this review is rather focused on AAV vector selection and their testing in experimental and preclinical NDD models. Preclinical and in vitro data reveal the strong potential of AAV for NDD-related diagnostics and therapeutic strategies.

Protein trans-splicing as a protein ligation tool to study protein structure and function

2011 ◽  
Vol 2 (3) ◽  
pp. 183-198 ◽  
Author(s):  
A. Sesilja Aranko ◽  
Gerrit Volkmann
Keyword(s):  
Protein Structure ◽  
Isotope Labeling ◽  
Successful Implementation ◽  
Protein Ligation ◽  
Current State ◽  
Split Intein ◽  
Segmental Isotope Labeling ◽  
Trans Splicing ◽  
And Function ◽  
Protein Reconstitution

AbstractProtein trans-splicing (PTS) exerted by split inteins is a protein ligation reaction which enables overcoming the barriers of conventional heterologous protein production. We provide an overview of the current state-of-the-art in split intein engineering, as well as the achievements of PTS technology in the realm of protein structure-function analyses, including incorporation of natural and artificial protein modifications, controllable protein reconstitution, segmental isotope labeling and protein cyclization. We further discuss factors crucial for the successful implementation of PTS in these protein engineering approaches, and speculate on necessary future endeavours to make PTS a universally applicable protein ligation tool.

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