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AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models.

Vasireddy V, Mills JA, Gaddameedi R, Basner-Tschakarjan E, Kohnke M, Black AD, Alexandrov K, Zhou S, Maguire AM, Chung DC, Mac H, Sullivan L, Gadue P, Bennicelli JL, French DL, Bennett J - PLoS ONE (2013)

Bottom Line: The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified.Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking.The gene transfer is efficient and the preliminary safety data are encouraging.

View Article: PubMed Central - PubMed

Affiliation: F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.

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Related in: MedlinePlus

Generation and Characterization of AAV2. hCHM.I). Schematic of the AAV proviral plasmid carrying human CHM. under the control of the cytomegalovirus enhancer chicken beta actin (eCBA) promoter. ITR: Inverted terminal repeats; Ori: Replication origin; KanR: Kanamycin resistance gene. II) i) Immunoblot and ii) fluorescent analysis reveals REP-1 protein in CHO cells transfected with pAAV2.hCHM. Lane A: Transfected cell (25 ug protein), B: Control (untransfected) cells, C- protein marker. Immunocytochemical analysis revealed the localization of REP-1 to the cytosolic region (II-ii-B; Green). No REP1 is observed in control cells (II-ii-A). Nuclei are stained with DAPI and appear blue. Scale bar is 50 uM. III) Immunoblot analysis of CHO cells infected with 1E3-2E5 viral genomes (vg) of AAV2. hCHM show an increase in REP-1 protein (indicated by arrow) proportional to the titer. Positive (+ve) control: pAAV2. hCHM-transfected CHO cell lysate.
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pone-0061396-g001: Generation and Characterization of AAV2. hCHM.I). Schematic of the AAV proviral plasmid carrying human CHM. under the control of the cytomegalovirus enhancer chicken beta actin (eCBA) promoter. ITR: Inverted terminal repeats; Ori: Replication origin; KanR: Kanamycin resistance gene. II) i) Immunoblot and ii) fluorescent analysis reveals REP-1 protein in CHO cells transfected with pAAV2.hCHM. Lane A: Transfected cell (25 ug protein), B: Control (untransfected) cells, C- protein marker. Immunocytochemical analysis revealed the localization of REP-1 to the cytosolic region (II-ii-B; Green). No REP1 is observed in control cells (II-ii-A). Nuclei are stained with DAPI and appear blue. Scale bar is 50 uM. III) Immunoblot analysis of CHO cells infected with 1E3-2E5 viral genomes (vg) of AAV2. hCHM show an increase in REP-1 protein (indicated by arrow) proportional to the titer. Positive (+ve) control: pAAV2. hCHM-transfected CHO cell lysate.

Mentions: Proviral plasmid pAAV2.CBAe.hCHM was generated and features wild type human CHM cDNA (hCHM) under the control of a hybrid, cytomegalovirus enhancer-chicken beta actin promoter (CBAe) (figure 1–I). The plasmid includes a kanamycin resistance gene and a 5.1 kb stuffer sequence to prevent reverse packaging of non-transgene containing sequence from the plasmid backbone. Sequence analyses confirmed the absence of potential mutations introduced during cloning. This plasmid will be available to investigators for the purpose of academic, non-commercial research in adherence to PLOS ONE policies, but also in a manner that is consistent with and subjugate to the most current US FDA regulations.


AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models.

Vasireddy V, Mills JA, Gaddameedi R, Basner-Tschakarjan E, Kohnke M, Black AD, Alexandrov K, Zhou S, Maguire AM, Chung DC, Mac H, Sullivan L, Gadue P, Bennicelli JL, French DL, Bennett J - PLoS ONE (2013)

Generation and Characterization of AAV2. hCHM.I). Schematic of the AAV proviral plasmid carrying human CHM. under the control of the cytomegalovirus enhancer chicken beta actin (eCBA) promoter. ITR: Inverted terminal repeats; Ori: Replication origin; KanR: Kanamycin resistance gene. II) i) Immunoblot and ii) fluorescent analysis reveals REP-1 protein in CHO cells transfected with pAAV2.hCHM. Lane A: Transfected cell (25 ug protein), B: Control (untransfected) cells, C- protein marker. Immunocytochemical analysis revealed the localization of REP-1 to the cytosolic region (II-ii-B; Green). No REP1 is observed in control cells (II-ii-A). Nuclei are stained with DAPI and appear blue. Scale bar is 50 uM. III) Immunoblot analysis of CHO cells infected with 1E3-2E5 viral genomes (vg) of AAV2. hCHM show an increase in REP-1 protein (indicated by arrow) proportional to the titer. Positive (+ve) control: pAAV2. hCHM-transfected CHO cell lysate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646845&req=5

pone-0061396-g001: Generation and Characterization of AAV2. hCHM.I). Schematic of the AAV proviral plasmid carrying human CHM. under the control of the cytomegalovirus enhancer chicken beta actin (eCBA) promoter. ITR: Inverted terminal repeats; Ori: Replication origin; KanR: Kanamycin resistance gene. II) i) Immunoblot and ii) fluorescent analysis reveals REP-1 protein in CHO cells transfected with pAAV2.hCHM. Lane A: Transfected cell (25 ug protein), B: Control (untransfected) cells, C- protein marker. Immunocytochemical analysis revealed the localization of REP-1 to the cytosolic region (II-ii-B; Green). No REP1 is observed in control cells (II-ii-A). Nuclei are stained with DAPI and appear blue. Scale bar is 50 uM. III) Immunoblot analysis of CHO cells infected with 1E3-2E5 viral genomes (vg) of AAV2. hCHM show an increase in REP-1 protein (indicated by arrow) proportional to the titer. Positive (+ve) control: pAAV2. hCHM-transfected CHO cell lysate.
Mentions: Proviral plasmid pAAV2.CBAe.hCHM was generated and features wild type human CHM cDNA (hCHM) under the control of a hybrid, cytomegalovirus enhancer-chicken beta actin promoter (CBAe) (figure 1–I). The plasmid includes a kanamycin resistance gene and a 5.1 kb stuffer sequence to prevent reverse packaging of non-transgene containing sequence from the plasmid backbone. Sequence analyses confirmed the absence of potential mutations introduced during cloning. This plasmid will be available to investigators for the purpose of academic, non-commercial research in adherence to PLOS ONE policies, but also in a manner that is consistent with and subjugate to the most current US FDA regulations.

Bottom Line: The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified.Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking.The gene transfer is efficient and the preliminary safety data are encouraging.

View Article: PubMed Central - PubMed

Affiliation: F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.

Show MeSH
Related in: MedlinePlus