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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

Trafficking of RAB27 protein is restored in affected cells after infection with AAV2.hCHM. CPF1 fibroblasts (i–vi) or CPS1 iPSCs (vii–xii) derived from CHM individuals showed improved trafficking of RAB27 after infection with AAV2. hCHM. In control CPF1 (i–iii) or CPS1 (vii–ix) untreated cells, Rab 27a (Green) was localized near the nucleus, whereas infection with AAV2. hCHM favored trafficking of RAB27 out of the perinuclear region in both CPF1(Rep-1 red, RAB27-green) (v–vi) and CPS1 (xi–xii) cells (REP1-green; RAB27-red). Nuclei are stained with DAPI and appear blue. II). Quantitative analysis of REP-1 and RAB27 levels in CHM iPSCs measured with imageStream. Histograms represent the increased level of exogenous REP-1 in cells infected with AAV2. hCHM (B) compared to controls (A) and unaffected wt controls (C). However, the level of REP-1 in transduced cells is reduced compared to unaffected control cells (E). Labeled Rab was increased in the surface mask in transduced cells (E) compared to uninfected cells (D). The levels of membrane-associated Rab 27 are comparable to the levels observed in unaffected wild type controls (F) Panel G and H shows representative cell images demonstrating the trafficking of Rab 27 to cell membrane in grey-scale. From left to right are shown: Brightfield, Rab 27, and REP-1, followed by composite images of REP-1 and RAB27. The cell surface masks used to define the inside and surface of the cell are overlayed in Brightfield and RAB/REP-1 labeled cells. White arrow in panel G, accumulated Rab inside the cell. Red arrowheads in panel H, presence of membrane Rab.
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pone-0061396-g005: Trafficking of RAB27 protein is restored in affected cells after infection with AAV2.hCHM. CPF1 fibroblasts (i–vi) or CPS1 iPSCs (vii–xii) derived from CHM individuals showed improved trafficking of RAB27 after infection with AAV2. hCHM. In control CPF1 (i–iii) or CPS1 (vii–ix) untreated cells, Rab 27a (Green) was localized near the nucleus, whereas infection with AAV2. hCHM favored trafficking of RAB27 out of the perinuclear region in both CPF1(Rep-1 red, RAB27-green) (v–vi) and CPS1 (xi–xii) cells (REP1-green; RAB27-red). Nuclei are stained with DAPI and appear blue. II). Quantitative analysis of REP-1 and RAB27 levels in CHM iPSCs measured with imageStream. Histograms represent the increased level of exogenous REP-1 in cells infected with AAV2. hCHM (B) compared to controls (A) and unaffected wt controls (C). However, the level of REP-1 in transduced cells is reduced compared to unaffected control cells (E). Labeled Rab was increased in the surface mask in transduced cells (E) compared to uninfected cells (D). The levels of membrane-associated Rab 27 are comparable to the levels observed in unaffected wild type controls (F) Panel G and H shows representative cell images demonstrating the trafficking of Rab 27 to cell membrane in grey-scale. From left to right are shown: Brightfield, Rab 27, and REP-1, followed by composite images of REP-1 and RAB27. The cell surface masks used to define the inside and surface of the cell are overlayed in Brightfield and RAB/REP-1 labeled cells. White arrow in panel G, accumulated Rab inside the cell. Red arrowheads in panel H, presence of membrane Rab.

Mentions: To determine whether infection of CHM cells with AAV2. hCHM corrects the primary protein trafficking defect which results from loss of REP-1 function, fibroblasts and iPSC lines were infected with AAV2. hCHM and assessed for changes in trafficking of RAB27, the major target of REP-1. Immunofluorescence analysis of control (untreated, affected) CPF1 and cells where REP-1 protein is absent (figure 5-I-i-iii,vii-ix), demonstrated the localization of RAB27 protein near the nuclear region (figure 5-I-ii-iii;viii-ix) whereas in presence of exogenous REP-1 (figure 5-I-iv-vi;x-xii), RAB27 was found to be trafficked to the membrane (figure 5-I-v-vi;xi-xii).


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)

Trafficking of RAB27 protein is restored in affected cells after infection with AAV2.hCHM. CPF1 fibroblasts (i–vi) or CPS1 iPSCs (vii–xii) derived from CHM individuals showed improved trafficking of RAB27 after infection with AAV2. hCHM. In control CPF1 (i–iii) or CPS1 (vii–ix) untreated cells, Rab 27a (Green) was localized near the nucleus, whereas infection with AAV2. hCHM favored trafficking of RAB27 out of the perinuclear region in both CPF1(Rep-1 red, RAB27-green) (v–vi) and CPS1 (xi–xii) cells (REP1-green; RAB27-red). Nuclei are stained with DAPI and appear blue. II). Quantitative analysis of REP-1 and RAB27 levels in CHM iPSCs measured with imageStream. Histograms represent the increased level of exogenous REP-1 in cells infected with AAV2. hCHM (B) compared to controls (A) and unaffected wt controls (C). However, the level of REP-1 in transduced cells is reduced compared to unaffected control cells (E). Labeled Rab was increased in the surface mask in transduced cells (E) compared to uninfected cells (D). The levels of membrane-associated Rab 27 are comparable to the levels observed in unaffected wild type controls (F) Panel G and H shows representative cell images demonstrating the trafficking of Rab 27 to cell membrane in grey-scale. From left to right are shown: Brightfield, Rab 27, and REP-1, followed by composite images of REP-1 and RAB27. The cell surface masks used to define the inside and surface of the cell are overlayed in Brightfield and RAB/REP-1 labeled cells. White arrow in panel G, accumulated Rab inside the cell. Red arrowheads in panel H, presence of membrane Rab.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0061396-g005: Trafficking of RAB27 protein is restored in affected cells after infection with AAV2.hCHM. CPF1 fibroblasts (i–vi) or CPS1 iPSCs (vii–xii) derived from CHM individuals showed improved trafficking of RAB27 after infection with AAV2. hCHM. In control CPF1 (i–iii) or CPS1 (vii–ix) untreated cells, Rab 27a (Green) was localized near the nucleus, whereas infection with AAV2. hCHM favored trafficking of RAB27 out of the perinuclear region in both CPF1(Rep-1 red, RAB27-green) (v–vi) and CPS1 (xi–xii) cells (REP1-green; RAB27-red). Nuclei are stained with DAPI and appear blue. II). Quantitative analysis of REP-1 and RAB27 levels in CHM iPSCs measured with imageStream. Histograms represent the increased level of exogenous REP-1 in cells infected with AAV2. hCHM (B) compared to controls (A) and unaffected wt controls (C). However, the level of REP-1 in transduced cells is reduced compared to unaffected control cells (E). Labeled Rab was increased in the surface mask in transduced cells (E) compared to uninfected cells (D). The levels of membrane-associated Rab 27 are comparable to the levels observed in unaffected wild type controls (F) Panel G and H shows representative cell images demonstrating the trafficking of Rab 27 to cell membrane in grey-scale. From left to right are shown: Brightfield, Rab 27, and REP-1, followed by composite images of REP-1 and RAB27. The cell surface masks used to define the inside and surface of the cell are overlayed in Brightfield and RAB/REP-1 labeled cells. White arrow in panel G, accumulated Rab inside the cell. Red arrowheads in panel H, presence of membrane Rab.
Mentions: To determine whether infection of CHM cells with AAV2. hCHM corrects the primary protein trafficking defect which results from loss of REP-1 function, fibroblasts and iPSC lines were infected with AAV2. hCHM and assessed for changes in trafficking of RAB27, the major target of REP-1. Immunofluorescence analysis of control (untreated, affected) CPF1 and cells where REP-1 protein is absent (figure 5-I-i-iii,vii-ix), demonstrated the localization of RAB27 protein near the nuclear region (figure 5-I-ii-iii;viii-ix) whereas in presence of exogenous REP-1 (figure 5-I-iv-vi;x-xii), RAB27 was found to be trafficked to the membrane (figure 5-I-v-vi;xi-xii).

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