The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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formulation and efficacy of eco prho abca4 sv40 nanoparticles for nonViral Gene Therapy of stargardt disease in a mouse model
Journal of Controlled Release, 2021Co-Authors: Da Sun, Wenyu Sun, Songqi Gao, Cheng Wei, Amirreza Naderi, Andrew L Schilb, Josef Scheidt, Sangjoon Lee, Timothy S Kern, Krzysztof PalczewskiAbstract:It is still a challenge to develop Gene replacement Therapy for retinal disorders caused by mutations in large Genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 Gene. Previously, we have developed an effective non-Viral Gene Therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced Gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal Gene Therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-Viral Gene Therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large Genes.
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targeted multifunctional lipid eco plasmid dna nanoparticles as efficient non Viral Gene Therapy for leber s congenital amaurosis
Molecular therapy. Nucleic acids, 2017Co-Authors: Da Sun, Songqi Gao, Bhubanananda Sahu, Rebecca M. Schur, Akiko Maeda, Amita Vaidya, Krzysztof PalczewskiAbstract:Development of a Gene delivery system with high efficiency and a good safety profile is essential for successful Gene Therapy. Here we developed a targeted non-Viral delivery system using a multifunctional lipid ECO for treating Leber’s congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high Gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65−/− mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-Viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2.
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Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-Viral Gene Therapy for Leber’s Congenital Amaurosis
Elsevier, 2017Co-Authors: Da Sun, Songqi Gao, Bhubanananda Sahu, Rebecca M. Schur, Amita M. Vaidya, Akiko Maeda, Krzysztof PalczewskiAbstract:Development of a Gene delivery system with high efficiency and a good safety profile is essential for successful Gene Therapy. Here we developed a targeted non-Viral delivery system using a multifunctional lipid ECO for treating Leber’s congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high Gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65−/− mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-Viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2
Da Sun - One of the best experts on this subject based on the ideXlab platform.
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formulation and efficacy of eco prho abca4 sv40 nanoparticles for nonViral Gene Therapy of stargardt disease in a mouse model
Journal of Controlled Release, 2021Co-Authors: Da Sun, Wenyu Sun, Songqi Gao, Cheng Wei, Amirreza Naderi, Andrew L Schilb, Josef Scheidt, Sangjoon Lee, Timothy S Kern, Krzysztof PalczewskiAbstract:It is still a challenge to develop Gene replacement Therapy for retinal disorders caused by mutations in large Genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 Gene. Previously, we have developed an effective non-Viral Gene Therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced Gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal Gene Therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-Viral Gene Therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large Genes.
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targeted multifunctional lipid eco plasmid dna nanoparticles as efficient non Viral Gene Therapy for leber s congenital amaurosis
Molecular therapy. Nucleic acids, 2017Co-Authors: Da Sun, Songqi Gao, Bhubanananda Sahu, Rebecca M. Schur, Akiko Maeda, Amita Vaidya, Krzysztof PalczewskiAbstract:Development of a Gene delivery system with high efficiency and a good safety profile is essential for successful Gene Therapy. Here we developed a targeted non-Viral delivery system using a multifunctional lipid ECO for treating Leber’s congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high Gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65−/− mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-Viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2.
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Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-Viral Gene Therapy for Leber’s Congenital Amaurosis
Elsevier, 2017Co-Authors: Da Sun, Songqi Gao, Bhubanananda Sahu, Rebecca M. Schur, Amita M. Vaidya, Akiko Maeda, Krzysztof PalczewskiAbstract:Development of a Gene delivery system with high efficiency and a good safety profile is essential for successful Gene Therapy. Here we developed a targeted non-Viral delivery system using a multifunctional lipid ECO for treating Leber’s congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high Gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65−/− mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-Viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2
Songqi Gao - One of the best experts on this subject based on the ideXlab platform.
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formulation and efficacy of eco prho abca4 sv40 nanoparticles for nonViral Gene Therapy of stargardt disease in a mouse model
Journal of Controlled Release, 2021Co-Authors: Da Sun, Wenyu Sun, Songqi Gao, Cheng Wei, Amirreza Naderi, Andrew L Schilb, Josef Scheidt, Sangjoon Lee, Timothy S Kern, Krzysztof PalczewskiAbstract:It is still a challenge to develop Gene replacement Therapy for retinal disorders caused by mutations in large Genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 Gene. Previously, we have developed an effective non-Viral Gene Therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced Gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal Gene Therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-Viral Gene Therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large Genes.
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targeted multifunctional lipid eco plasmid dna nanoparticles as efficient non Viral Gene Therapy for leber s congenital amaurosis
Molecular therapy. Nucleic acids, 2017Co-Authors: Da Sun, Songqi Gao, Bhubanananda Sahu, Rebecca M. Schur, Akiko Maeda, Amita Vaidya, Krzysztof PalczewskiAbstract:Development of a Gene delivery system with high efficiency and a good safety profile is essential for successful Gene Therapy. Here we developed a targeted non-Viral delivery system using a multifunctional lipid ECO for treating Leber’s congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high Gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65−/− mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-Viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2.
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Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-Viral Gene Therapy for Leber’s Congenital Amaurosis
Elsevier, 2017Co-Authors: Da Sun, Songqi Gao, Bhubanananda Sahu, Rebecca M. Schur, Amita M. Vaidya, Akiko Maeda, Krzysztof PalczewskiAbstract:Development of a Gene delivery system with high efficiency and a good safety profile is essential for successful Gene Therapy. Here we developed a targeted non-Viral delivery system using a multifunctional lipid ECO for treating Leber’s congenital amaurosis type 2 (LCA2) and tested this in a mouse model. ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low amine to phosphate (N/P) ratio and mediated high Gene transfection efficiency in ARPE-19 cells because of their intrinsic properties of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC). All-trans-retinylamine, which binds to interphotoreceptor retinoid-binding protein (IRBP), was incorporated into the nanoparticles via a polyethylene glycol (PEG) spacer for targeted delivery of pDNA into the retinal pigmented epithelium. The targeted ECO/pDNA nanoparticles provided high GFP expression in the RPE of 1-month-old Rpe65−/− mice after subretinal injection. Such mice also exhibited a significant increase in electroretinographic activity, and this therapeutic effect continued for at least 120 days. A safety study in wild-type BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles. All-trans-retinylamine-modified ECO/pDNA nanoparticles provide a promising non-Viral platform for safe and effective treatment of RPE-specific monogenic eye diseases such as LCA2
Claudia Wehrspaun - One of the best experts on this subject based on the ideXlab platform.
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high throughput 5 utr engineering for enhanced protein production in non Viral Gene therapies
Nature Communications, 2021Co-Authors: Jicong Cao, Eva Maria Novoa, Zhizhuo Zhang, William C W Chen, Dianbo Liu, Gigi C G Choi, Alan S L Wong, Claudia WehrspaunAbstract:Despite significant clinical progress in cell and Gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5' UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5' UTRs with high translation efficiencies and use this information with in silico Genetic algorithms to Generate synthetic 5' UTRs. A total of ~12,000 5' UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiViral approaches. Using this approach, we identify three synthetic 5' UTRs that outperform commonly used non-Viral Gene Therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5' UTR libraries with recombinase-mediated integration can identify Genetic elements that enhance protein expression, which should have numerous applications for engineered cell and Gene therapies.
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high throughput 5 utr engineering for enhanced protein production in non Viral Gene therapies
bioRxiv, 2020Co-Authors: Jicong Cao, Eva Maria Novoa, Zhizhuo Zhang, William C W Chen, Dianbo Liu, Gigi C G Choi, Alan S L Wong, Claudia Wehrspaun, Manolis KellisAbstract:Despite significant clinical progress in cell and Gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5′ untranslated regions (5′ UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5′UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5′ UTRs with high translation efficiencies and used this information with in silico Genetic algorithms to Generate synthetic 5′ UTRs. A total of ~12,000 5′ UTRs were then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiViral approaches. Using this approach, we identified three synthetic 5′ UTRs that outperformed commonly used non-Viral Gene Therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5′ UTRs enabled even higher protein expression than obtained with each individual 5′ UTR. In summary, we demonstrate that high-throughput screening of 5′ UTR libraries with recombinase-mediated integration can identify Genetic elements that enhance protein expression, which should have numerous applications for engineered cell and Gene therapies.
Cyrus R Safinya - One of the best experts on this subject based on the ideXlab platform.
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cationic lipid dna complexes for non Viral Gene Therapy relating supramolecular structures to cellular pathways
Expert Opinion on Biological Therapy, 2005Co-Authors: Kai K Ewert, Ayesha Ahmad, Heather M Evans, Cyrus R SafinyaAbstract:Cationic liposomes (CLs) are used as nonViral vectors in worldwide human clinical trials of Gene Therapy. Among other advantages, lipid–DNA complexes have the ability to transfer very large Genes into cells, but their efficiency is much lower than that of viruses. Recent studies combining structural and biological techniques are beginning to unravel the relationship between the distinctly structured CL–DNA complexes and their transfection efficiency. Most CL–DNA complexes form a multilayered structure with DNA sandwiched between the cationic lipids (lamellar complexes, LαC). On rare occasions, an inverted hexagonal structure (HIIC) is observed. An important recent insight is that the membrane charge density (σM) of the CL-vector is a universal parameter governing the transfection efficiency of LαC (but not HIIC) complexes. This has led to a new model of the cellular uptake of LαC complexes through activated fusion with endosomal membranes. Surface-functionalised complexes with poly(ethylene glycol)-lipids...
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cationic lipid dna complexes for Gene Therapy understanding the relationship between complex structure and Gene delivery pathways at the molecular level
Current Medicinal Chemistry, 2004Co-Authors: Kai K Ewert, Ayesha Ahmad, Heather M Evans, Nelle L Slack, Alison J Lin, Charles E Samuel, Cyrus R SafinyaAbstract:Cationic liposomes (CLs) are used as Gene vectors (carriers) in worldwide human clinical trials of non-Viral Gene Therapy. These lipid-Gene complexes have the potential of transferring large pieces of DNA of up to 1 million base-pairs into cells. As our understanding of the mechanisms of action of CL-DNA complexes remains poor, transfection efficiencies are still low when compared to Gene delivery with Viral vectors. We describe recent studies with a combination of techniques (synchrotron x-ray diffraction for structure determination, laser-scanning confocal microscopy to probe the interactions of CL-DNA particles with cells, and luciferase reporter-Gene expression assays to measure transfection efficiencies in mammalian cells), which collectively are beginning to unravel the relationship between the distinctly structured CL-DNA complexes and their transfection efficiency. The work described here is applicable to transfection optimization in ex vivo cell transfection, where cells are removed and returned to patients after transfection. CL-DNA complexes primarily form a multilayered sandwich structure with DNA layered between the cationic lipids (labeled L(alpha)(C)). On rare occasions, an inverted hexagonal structure with DNA encapsulated in lipid tubules (labeled H(II)(C)) is observed. A major recent insight is that for L(alpha)(C) complexes the membrane charge density sigma(M) of the CL-vector, rather than the charge of the cationic lipid alone, is a key universal parameter that governs the transfection efficiency of L(alpha)(C) complexes in cells. The parameter sigma(M) is a measure of the average charge per unit area of the membrane, thus taking into account the amount of neutral lipids. In contrast to L(alpha)(C) complexes, H(II)(C) complexes containing the lipid 1,2-dioleoyl-sn-glycerophosphatidylethanolamine (DOPE) exhibit no dependence on sigma(M). The current limiting factor to transfection by cationic lipid vectors appears to be the tight association of a fraction of the delivered exogenous DNA with cationic cellular molecules, which may prevent optimal transcriptional activity. Future directions are outlined, which make use of surface-functionalized CL-DNA complexes suitable for transfection in vivo.
