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Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells
EURURO-6489; No. of Pages 3 EUROPEAN UROLOGY XXX (2015) XXX–XXX
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Research Letter
Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells Jianzhong Ai a,b,y, Dan Wang b,y, Qiang Wei a,y, Hong Li a,*, Guangping Gao b,c,d,* Prostate diseases can severely compromise the lifespan and quality of life of males, yet highly effective clinical interventions for prostate diseases are still lacking. As the genetic basis of prostate diseases was gradually unraveled, gene therapy was explored as a therapeutic strategy for prostate diseases [1]. Recombinant adeno-associated virus (rAAV) vectors possess many advantages in gene therapy applications, including low immunogenicity and genotoxicity, broad tissue tropism and high transduction efficiency in vivo, and long-term transgene expression [2,3]. The aim of this study was to identify rAAV serotypes that can efficiently transduce prostate tissue and prostate cancer cells, and could thus pave the way for rAAV-based gene therapy targeting prostate diseases. We directly injected rAAV vectors of 12 serotypes expressing enhanced green fluorescent protein (EGFP) into wild-type mouse prostate comprising anterior prostate (AP) and dorsal lateral prostate (DLP). At 3 wk after injection, EGFP fluorescence in AP and DLP cryosections was quantified to assess transduction efficiency. We found that rAAV6.2 and rAAV7 outperformed the other serotypes in transducing the whole prostate including AP and DLP (Fig. 1A,B). For these two leading serotypes, we determined that the vector genome biodistribution in injected AP and DLP was approximately 10–20 rAAV genome copies per diploid genome (Fig. 1C). Moreover, rAAV6.2 and rAAV7 were able to transduce 65–80% of luminal, basal, and stromal cells in AP and DLP (Fig. 1D,E). Luminal and basal cells are considered as the origin of prostate cancer [4,5], and stromal cells are the primary cell type affected in
benign prostate hyperplasia. Therefore, efficient transduction of these cell types by rAAV6.2 and rAAV7 warrants further development of gene therapy as a preventive and/or curative treatment for various prostate diseases. We did not observe any adverse pathologic change in AP or DLP according to hematoxylin and eosin staining after rAAV injection (data not shown). Our results suggest that intraprostate injection of rAAV6.2 and rAAV7 is an efficient and safe means for delivery of genes of interest to mouse prostate in vivo. In addition, we reasoned that identification of rAAV serotypes that efficiently transduce prostate cancer cells in vitro could provide guidance for gene delivery into prostate cancer xenografts in vivo, a potential therapeutic strategy for prostate cancer. To this end, using fluorescence microscopy and flow cytometry we identified rAAV5, rAAV6, and rAAV6.2 as the leading serotypes for in vivo transduction of both PC3 and TRAMP-C2 cells, representing human and mouse prostate cancer cells, respectively (data not shown). In summary, we carried out an extensive characterization of major rAAV serotype vectors available for transducing prostate tissue and cells for the first time, and discovered that several rAAV serotypes could efficiently transduce normal prostate tissue in vivo and prostate cancer cells in vitro. These findings establish a critical foundation for further development of rAAV-based gene therapy strategies for prostate diseases, and provide a powerful in vitro and in vivo research tool for studying prostate diseases.
http://dx.doi.org/10.1016/j.eururo.2015.10.019 0302-2838/Published by Elsevier B.V. on behalf of European Association of Urology.
Please cite this article in press as: Ai J, et al. Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.10.019
EURURO-6489; No. of Pages 3 2
EUROPEAN UROLOGY XXX (2015) XXX–XXX
Fig. 1 – rAAV6.2 and rAAV7 efficiently transduced mouse prostate following intraprostate injection. (A) Quantification of transduction efficiency in AP (yellow bars) and DLP (orange bars) following intraprostate injection with rAAV vectors of different serotypes expressing EGFP. EGFP fluorescence intensity of cryosections is presented in arbitrary units (a.u.) (B) Representative fluorescence images of AP and DLP cryosections showing merger of EGFP native fluorescence (green) and nuclear staining by DAPI (blue) following injection of PBS, rAAV6.2, or rAAV7. Scale bars, 25 mm. (C) Biodistribution of rAAV genomes in AP (yellow bars) and DLP (orange bars) following intraprostate injection of rAAV6.2 and rAAV7. Data are presented as rAAV genome copies per diploid genome. (D) Representative images of immunofluorescence staining of prostate luminal cells (top panels), basal cells (middle panels), and stromal cells (bottom panels), marked by K8, K5, and a-actin staining, respectively. Native EGFP fluorescence images and merged images with DAPI staining (blue) for the same sections are also shown. Arrows indicate representative co-localization of EGFP and cell-type marker signals. (E) Quantification of the percentage of EGFP-positive cells for each cell type. rAAV = recombinant adeno-associated virus; AP = anterior prostate; DLP = dorsal lateral prostate; EGFP = enhanced green fluorescent protein; DAPI = 40 ,6-diamidino-2-phenylindole.
Conflicts of interest: Guangping Gao is a co-founder of Voyager
(‘‘863’’ Program) of China (2012AA020810) to G.G., an internal grant
Therapeutics and holds equity in the company, and is an inventor listed
from Sichuan University to H.L., and a grant from the Project Funded by
on patents with potential royalties licensed to Voyager Therapeutics and
China Postdoctoral Science Foundation (2015T80980) to J.A.
other biopharmaceutical companies. The remaining authors have nothing to disclose.
References [1] Dash R, Azab B, Shen XN, et al. Developing an effective gene
Acknowledgments: This work was supported by internal grants from the
therapy for prostate cancer: new technologies with potential to
University of Massachusetts Medical School and a grant from the
translate from the laboratory into the clinic. Discov Med 2011;11:
National High Technology Research and Development Program
46–56.
Please cite this article in press as: Ai J, et al. Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.10.019
EURURO-6489; No. of Pages 3 3
EUROPEAN UROLOGY XXX (2015) XXX–XXX
[2] Wang D, Gao G. State-of-the-art human gene therapy: part I. Gene
c
Department of Microbiology and Physiology Systems, University of
delivery technologies. Discov Med 2014;18:67–77.
Massachusetts Medical School, Worcester, MA, USA
[3] Wang D, Gao G. State-of-the-art human gene therapy: part II. Gene
d
State Key Laboratory of Biotherapy, West China Hospital,
therapy strategies and clinical applications. Discov Med 2014;18:
Sichuan University, Chengdu, P.R. China
151–61. [4] Goldstein AS, Huang J, Guo C, Garraway IP, Witte ON. Identification of a cell of origin for human prostate cancer. Science 2010;329:
*Corresponding authors. Horae Gene Therapy Center, University of Massachusetts Medical School, 368 Plantation Street, AS6-2049, Worcester, MA 01605, USA. Tel. +1 508 8563563; Fax: +1 508 8561552.
568–71. [5] Wang X, Kruithof-de Julio M, Economides KD, et al. A luminal
Department of Urology, West China Hospital, Sichuan University,
epithelial stem cell that is a cell of origin for prostate cancer. Nature
Guoxue Xiang #37, Chengdu, Sichuan 610041, P.R. China. Tel. +86 28 85125449; Fax: +86-28-85422444.
2009;461:495–500.
E-mail addresses: [email protected] (H. Li), a
[email protected] (G. Gao).
Department of Urology, West China Hospital, Sichuan University, Chengdu, P.R. China
y
These authors contributed equally to this work.
b
Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA
October 6, 2015
Please cite this article in press as: Ai J, et al. Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.10.019
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Research Letter
Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells Jianzhong Ai a,b,y, Dan Wang b,y, Qiang Wei a,y, Hong Li a,*, Guangping Gao b,c,d,* Prostate diseases can severely compromise the lifespan and quality of life of males, yet highly effective clinical interventions for prostate diseases are still lacking. As the genetic basis of prostate diseases was gradually unraveled, gene therapy was explored as a therapeutic strategy for prostate diseases [1]. Recombinant adeno-associated virus (rAAV) vectors possess many advantages in gene therapy applications, including low immunogenicity and genotoxicity, broad tissue tropism and high transduction efficiency in vivo, and long-term transgene expression [2,3]. The aim of this study was to identify rAAV serotypes that can efficiently transduce prostate tissue and prostate cancer cells, and could thus pave the way for rAAV-based gene therapy targeting prostate diseases. We directly injected rAAV vectors of 12 serotypes expressing enhanced green fluorescent protein (EGFP) into wild-type mouse prostate comprising anterior prostate (AP) and dorsal lateral prostate (DLP). At 3 wk after injection, EGFP fluorescence in AP and DLP cryosections was quantified to assess transduction efficiency. We found that rAAV6.2 and rAAV7 outperformed the other serotypes in transducing the whole prostate including AP and DLP (Fig. 1A,B). For these two leading serotypes, we determined that the vector genome biodistribution in injected AP and DLP was approximately 10–20 rAAV genome copies per diploid genome (Fig. 1C). Moreover, rAAV6.2 and rAAV7 were able to transduce 65–80% of luminal, basal, and stromal cells in AP and DLP (Fig. 1D,E). Luminal and basal cells are considered as the origin of prostate cancer [4,5], and stromal cells are the primary cell type affected in
benign prostate hyperplasia. Therefore, efficient transduction of these cell types by rAAV6.2 and rAAV7 warrants further development of gene therapy as a preventive and/or curative treatment for various prostate diseases. We did not observe any adverse pathologic change in AP or DLP according to hematoxylin and eosin staining after rAAV injection (data not shown). Our results suggest that intraprostate injection of rAAV6.2 and rAAV7 is an efficient and safe means for delivery of genes of interest to mouse prostate in vivo. In addition, we reasoned that identification of rAAV serotypes that efficiently transduce prostate cancer cells in vitro could provide guidance for gene delivery into prostate cancer xenografts in vivo, a potential therapeutic strategy for prostate cancer. To this end, using fluorescence microscopy and flow cytometry we identified rAAV5, rAAV6, and rAAV6.2 as the leading serotypes for in vivo transduction of both PC3 and TRAMP-C2 cells, representing human and mouse prostate cancer cells, respectively (data not shown). In summary, we carried out an extensive characterization of major rAAV serotype vectors available for transducing prostate tissue and cells for the first time, and discovered that several rAAV serotypes could efficiently transduce normal prostate tissue in vivo and prostate cancer cells in vitro. These findings establish a critical foundation for further development of rAAV-based gene therapy strategies for prostate diseases, and provide a powerful in vitro and in vivo research tool for studying prostate diseases.
http://dx.doi.org/10.1016/j.eururo.2015.10.019 0302-2838/Published by Elsevier B.V. on behalf of European Association of Urology.
Please cite this article in press as: Ai J, et al. Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.10.019
EURURO-6489; No. of Pages 3 2
EUROPEAN UROLOGY XXX (2015) XXX–XXX
Fig. 1 – rAAV6.2 and rAAV7 efficiently transduced mouse prostate following intraprostate injection. (A) Quantification of transduction efficiency in AP (yellow bars) and DLP (orange bars) following intraprostate injection with rAAV vectors of different serotypes expressing EGFP. EGFP fluorescence intensity of cryosections is presented in arbitrary units (a.u.) (B) Representative fluorescence images of AP and DLP cryosections showing merger of EGFP native fluorescence (green) and nuclear staining by DAPI (blue) following injection of PBS, rAAV6.2, or rAAV7. Scale bars, 25 mm. (C) Biodistribution of rAAV genomes in AP (yellow bars) and DLP (orange bars) following intraprostate injection of rAAV6.2 and rAAV7. Data are presented as rAAV genome copies per diploid genome. (D) Representative images of immunofluorescence staining of prostate luminal cells (top panels), basal cells (middle panels), and stromal cells (bottom panels), marked by K8, K5, and a-actin staining, respectively. Native EGFP fluorescence images and merged images with DAPI staining (blue) for the same sections are also shown. Arrows indicate representative co-localization of EGFP and cell-type marker signals. (E) Quantification of the percentage of EGFP-positive cells for each cell type. rAAV = recombinant adeno-associated virus; AP = anterior prostate; DLP = dorsal lateral prostate; EGFP = enhanced green fluorescent protein; DAPI = 40 ,6-diamidino-2-phenylindole.
Conflicts of interest: Guangping Gao is a co-founder of Voyager
(‘‘863’’ Program) of China (2012AA020810) to G.G., an internal grant
Therapeutics and holds equity in the company, and is an inventor listed
from Sichuan University to H.L., and a grant from the Project Funded by
on patents with potential royalties licensed to Voyager Therapeutics and
China Postdoctoral Science Foundation (2015T80980) to J.A.
other biopharmaceutical companies. The remaining authors have nothing to disclose.
References [1] Dash R, Azab B, Shen XN, et al. Developing an effective gene
Acknowledgments: This work was supported by internal grants from the
therapy for prostate cancer: new technologies with potential to
University of Massachusetts Medical School and a grant from the
translate from the laboratory into the clinic. Discov Med 2011;11:
National High Technology Research and Development Program
46–56.
Please cite this article in press as: Ai J, et al. Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.10.019
EURURO-6489; No. of Pages 3 3
EUROPEAN UROLOGY XXX (2015) XXX–XXX
[2] Wang D, Gao G. State-of-the-art human gene therapy: part I. Gene
c
Department of Microbiology and Physiology Systems, University of
delivery technologies. Discov Med 2014;18:67–77.
Massachusetts Medical School, Worcester, MA, USA
[3] Wang D, Gao G. State-of-the-art human gene therapy: part II. Gene
d
State Key Laboratory of Biotherapy, West China Hospital,
therapy strategies and clinical applications. Discov Med 2014;18:
Sichuan University, Chengdu, P.R. China
151–61. [4] Goldstein AS, Huang J, Guo C, Garraway IP, Witte ON. Identification of a cell of origin for human prostate cancer. Science 2010;329:
*Corresponding authors. Horae Gene Therapy Center, University of Massachusetts Medical School, 368 Plantation Street, AS6-2049, Worcester, MA 01605, USA. Tel. +1 508 8563563; Fax: +1 508 8561552.
568–71. [5] Wang X, Kruithof-de Julio M, Economides KD, et al. A luminal
Department of Urology, West China Hospital, Sichuan University,
epithelial stem cell that is a cell of origin for prostate cancer. Nature
Guoxue Xiang #37, Chengdu, Sichuan 610041, P.R. China. Tel. +86 28 85125449; Fax: +86-28-85422444.
2009;461:495–500.
E-mail addresses: [email protected] (H. Li), a
[email protected] (G. Gao).
Department of Urology, West China Hospital, Sichuan University, Chengdu, P.R. China
y
These authors contributed equally to this work.
b
Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA, USA
October 6, 2015
Please cite this article in press as: Ai J, et al. Adeno-associated Virus Serotype Vectors Efficiently Transduce Normal Prostate Tissue and Prostate Cancer Cells. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.10.019