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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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Prenylation activity in fibroblasts (CPF1) (i) and iPSCs (CPS1, CPS2) (ii) cultured from CHM individuals is restored following infection with AAV2.hCHM. Prenylation assay was performed using the cytosolic fraction of cells transduced with AAV2. hCHM and from untreated affected cells (Control). Cell lysates were incubated with RabGGTase, RAB27 and [3H]-labeled GGPP. A significant increase (P<0.02) in the prenylation activity of exogenous REP-1 was observed in both CPF1 (2 fold) and CPS1, CPS2 cells (∼3 fold).
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pone-0061396-g004: Prenylation activity in fibroblasts (CPF1) (i) and iPSCs (CPS1, CPS2) (ii) cultured from CHM individuals is restored following infection with AAV2.hCHM. Prenylation assay was performed using the cytosolic fraction of cells transduced with AAV2. hCHM and from untreated affected cells (Control). Cell lysates were incubated with RabGGTase, RAB27 and [3H]-labeled GGPP. A significant increase (P<0.02) in the prenylation activity of exogenous REP-1 was observed in both CPF1 (2 fold) and CPS1, CPS2 cells (∼3 fold).

Mentions: To determine whether infection with AAV2. hCHM restores REP-1 function in cells of patients with loss of function CHM mutations, CPF1, CPS1, and CPS2 cells were infected with AAV2. hCHM at an MOI of 2×105. Forty-eight hours post infection, cells were harvested and cytosolic fractions of the cells were isolated and used to perform in vitro prenylation assays in which the cytosolic cellular fraction served as the REP-1 protein source, RabGGTase, [3H]-GGPP as the prenyl group donor, and recombinant RAB27 as the substrate, as described previously [30], [38], [39]. We observed a significant increase in the prenylation of RAB27a in the fibroblast line, CPF1 (∼2 fold, 3-i; p = 0.01) and the iPSC lines, CPS1 and CPS2 (∼3 fold, figure 3-ii; p = 0.0015) cells transduced with AAV2. hCHM compared to the controls (figure 4-i,ii).


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)

Prenylation activity in fibroblasts (CPF1) (i) and iPSCs (CPS1, CPS2) (ii) cultured from CHM individuals is restored following infection with AAV2.hCHM. Prenylation assay was performed using the cytosolic fraction of cells transduced with AAV2. hCHM and from untreated affected cells (Control). Cell lysates were incubated with RabGGTase, RAB27 and [3H]-labeled GGPP. A significant increase (P<0.02) in the prenylation activity of exogenous REP-1 was observed in both CPF1 (2 fold) and CPS1, CPS2 cells (∼3 fold).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0061396-g004: Prenylation activity in fibroblasts (CPF1) (i) and iPSCs (CPS1, CPS2) (ii) cultured from CHM individuals is restored following infection with AAV2.hCHM. Prenylation assay was performed using the cytosolic fraction of cells transduced with AAV2. hCHM and from untreated affected cells (Control). Cell lysates were incubated with RabGGTase, RAB27 and [3H]-labeled GGPP. A significant increase (P<0.02) in the prenylation activity of exogenous REP-1 was observed in both CPF1 (2 fold) and CPS1, CPS2 cells (∼3 fold).
Mentions: To determine whether infection with AAV2. hCHM restores REP-1 function in cells of patients with loss of function CHM mutations, CPF1, CPS1, and CPS2 cells were infected with AAV2. hCHM at an MOI of 2×105. Forty-eight hours post infection, cells were harvested and cytosolic fractions of the cells were isolated and used to perform in vitro prenylation assays in which the cytosolic cellular fraction served as the REP-1 protein source, RabGGTase, [3H]-GGPP as the prenyl group donor, and recombinant RAB27 as the substrate, as described previously [30], [38], [39]. We observed a significant increase in the prenylation of RAB27a in the fibroblast line, CPF1 (∼2 fold, 3-i; p = 0.01) and the iPSC lines, CPS1 and CPS2 (∼3 fold, figure 3-ii; p = 0.0015) cells transduced with AAV2. hCHM compared to the controls (figure 4-i,ii).

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