Leopold Lab

Leopold Lab

Research Projects

  • Gene therapy - viral mimics
  • Host - virus interactions
  • Gene expression in human disease

Gene therapy – Viral mimics
In the world of gene therapy, the major strategies for accomplishing transfer of DNA to the nucleus of a cell involve either disabled viruses or chemical chaperones. Members of the former group are very efficient in terms of targeting their genetic payload to the nucleus, but elicit a specific immune response against the virus. Members of the latter group avoid a targeted immune response, but lack efficiency. Armed with an understanding of the mechanisms by which viral vectors, such as adenovirus, deliver DNA to the nucleus, we hypothesize that the efficiency of chemical vectors can be improved by mimicking viral mechanisms. To address this hypothesis, experiments are aimed at creating and assembling novel biochemical entities that will display the characteristics of viral proteins. The experimental approach includes evaluation of peptides, lipids, and carbohydrates, both novel and well known, using assembly methods inspired by viruses.

Evaluation of a novel lipid. The first step in cell entry involves crossing the plasma membrane. Working with a novel class of lipids developed by C. L. Oepstad, V. Partali, and R. Sliwka (Norwegian University of Science and Technology) and characterized by M. Pungente (Weill Cornell Medical College – Qatar), the ability of the lipid to deliver nucleic acid to the cytoplasm was evaluated by phase (left panel) and fluorescence (right panel) microscopy.

Review Articles

Leopold PL. 2000. Fluorescence methods reveal intracellular trafficking of gene transfer vectors: The light toward the end of the tunnel. Molec. Ther. 1:302-303.

Leopold PL. 2003. Cell physiology as a variable in gene transfer to endothelium. Curr. Atheroscler. Repts. 5:171-177.

Leopold PL, Pfister KK. 2006. Viral strategies for intracellular trafficking: motors and microtubules. Traffic 7:516-523.

Leopold PL. Chapter 34: Microtubule-dependent motility during intracellular trafficking of vector genome to the nucleus: Subcellular mimicry in virology and nanoengineering. In Nanotechnology in Biology and Medicine, T. Vo-Dinh, Ed., Taylor and Francis, Inc./CRC Press Inc., London, 2007.

Leopold PL, Crystal RG. 2007. Intracellular trafficking of adenovirus: Many means to many ends. Adv. Drug Deliv. Rev. 59:810-821.

Research Articles

Leopold PL, Ferris B, Grinberg I, Worgall S, Hackett NR, Crystal RG. 1998. Fluorescent virions: Dynamic tracking of adenovirus gene transfer vectors. Hum. Gene Ther. 9:367-378.

Hidaka C, Milano E, Leopold PL, Bergelson JM, Hackett NR, Finberg RW, Wickham TJ, Kovesdi I, Roelvink P, Warren RF, Crystal RG. 1999. CAR-dependent and CAR-independent pathways of adenovirus vector-mediated gene transfer and expression in primary human skin fibroblasts. J. Clin. Invest. 103:579-587.

Kaner RJ, Worgall S, Leopold PL, Stolze E, Milano E, Hidaka C, Ramalingam R, Hackett NR, Singh R, Bergelson J, Finberg R, Falck-Pedersen E, Crystal RG. 1999. Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor. Am. J. Respir. Cell Mol. Biol. 20:361-370.

Miyazawa N, Leopold PL, Hackett N, Ferris B, Worgall S, Falck-Pedersen E, Crystal RG. 1999. “Fiber swap” between adenovirus subgroups B and C alters intracellular trafficking of adenovirus gene transfer vectors. J. Virol. 73:6056-6065.

Wisnivesky JP, Leopold PL, Crystal RG. 1999. Specific binding of the adenovirus capsid to the nuclear envelope. Hum. Gene Ther. 10:2187-2195.

Leopold PL, Kreitzer G, Miyazawa N, Rempel S, Pfister KK, Rodriguez-Boulan E, Crystal RG. 2000. Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum. Gene Ther. 11:151-165.

Worgall S, Worgall TS, Kostarelos K, Singh R, Leopold PL, Hackett NR, Crystal RG. 2000. Free cholesterol enhances adenoviral vector gene transfer and expression in CAR-deficient cells. Molec. Ther. 1:39-48.

Miyazawa N, Crystal RG, Leopold PL. 2001. Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein. J. Virol. 75:1387-1400.

Vincent T, Harvey BG, Hogan SM, Bailey CJ, Crystal RG, Leopold PL. 2001. Rapid assessment of adenovirus serum neutralizing antibody titer based on a quantitative, morphometric evaluation of capsid binding and intracellular trafficking. J. Virol. 75:1516-1521.

Seidman MA, Hogan SM, Wendland RL, Worgall S, Crystal RG, Leopold PL. 2001. Variation in adenovirus receptor expression and adenovirus vector-mediated transgene expression at defined stages of the cell cycle. Molec. Ther. 4:13-21.

Bailey CJ, Crystal RG, Leopold PL. 2003. Association of adenovirus with the microtubule organizing center. J. Virol. 77:13275-13287.

Vincent T, Pettersson RF, Crystal RG, Leopold PL. 2004. Cytokine-mediated downregulation of coxsackie-adenovirus receptor in endothelial cells. J. Virol. 78:8047-58.

Kelkar S, Pfister KK, Crystal RG, Leopold PL. 2004. Cytoplasmic dynein mediates adenovirus binding to microtubules. J. Virol. 78:10122-10132.

Kelkar S, De B, Gao G, Wilson JM, Crystal RG, Leopold PL. 2006. A common mechanism for cytoplasmic dynein-dependent microtubule binding shared among adeno-associated virus and adenovirus serotypes. J. Virol. 80:7781-7785.

Leopold PL, Wendland R, Vincent T, Crystal RG. 2006. Neutralized adenovirus-immune complexes can mediate effective gene transfer via a Fc receptor-dependent infection pathway. J. Virol. 80:10237-10247.

Host-virus Interaction

Viral infection of host cells triggers a variety of cellular responses. Well known responses include the effects of viral proteins on host DNA transcription and translation as well as expression of cellular defense proteins such as interferon. Our hypothesis focuses on the early stages of infection with the hypothesis that viral capsids are “seen” by cells as misfolded proteins. Cells have mechanisms for responding to misfolded proteins ranging from targeted degradation to a global unfolded protein response. Experiments in this area will address the degree to which the cellular response is instrumental in viral infection as well as the potential consequences of that response to cell viability and function.

Common mechanism? Despite an absence of shared protein structures, the capsids of adenovirus and adeno-associated virus compete for binding with cytoplasmic dynein on microtubules. Is microtubule-based viral trafficking a general property of foreign proteins in the cytoplasm, or is it a specific property in each viral capsid? (Reproduced from Kelkar et al., 2006.)

Gene Expresssion in Human Disease
One of our long-standing interests relates to developing an understanding of human disease at the cellular and molecular level. Through a series of collaborations, a variety of human disease models have been examined. Human disease will be a continuing theme in the laboratory. Our disease-related research covers viral infection-immunity, cystic fibrosis and other pulmonary diseases, regenerative medicine, cancer, and lysosomal storage diseases. A list of prior disease-related research white papers follows.

Viral Infection/Immunity

Worgall S, Leopold PL, Wolff G, van Roijen N, Crystal RG. 1997. Role of alveolar macrophages in rapid elimination of adenovirus vectors administered to the epithelial surface of the respiratory tract. Hum. Gene Ther. 8:1675-1684.

Bezdicek P, Worgall S, Kovesdi I, Kim M-K, Park J-G, Vincent T, Leopold PL, Schreiber AD, Crystal RG. 1999. Enhanced liver uptake of opsonized red blood cells following in vivo transfer of Fc RII cDNA. Blood 94:3448-3455.

Harvey BG, Worgall S, Ely S, Leopold PL, Crystal RG. 1999. Cellular immune responses of healthy individuals to intradermal administration of an E1- E3- adenovirus gene transfer vector. Hum. Gene Ther. 10:2823-2837.

Vincent T, Harvey BG, Hogan SM, Bailey CJ, Crystal RG, Leopold PL. 2001. Rapid assessment of adenovirus serum neutralizing antibody titer based on a quantitative, morphometric evaluation of capsid binding and intracellular trafficking. J. Virol. 75:1516-1521.

Crystal RG, Harvey BG, Wisnivesky JP, O=Donoghue KA, Chu KW, Maroni J, Muscat JC, Pippo AL, Wright C, Kaner RJ, Leopold PL, Kessler P, Rasmussen H, Rosengart TK, Hollman C. 2002. Analysis of risk factors for local delivery of low and intermediate dose adenovirus gene transfer vectors to individuals with a spectrum of co-morbid conditions. Hum. Gene Ther. 13:65-100.

Rice J, Connor R, Worgall S, Moore JP, Leopold PL, Kaner RJ, Crystal RG. 2002. Inhibition of HIV-1 replication in alveolar macrophages by adenovirus gene transfer vectors. Am. J. Resp. Cell Molec. Biol. 27:214-219.

Worgall S, Busch A, Rivara M, Bonnyay D, Leopold PL, Merritt R, Hackett NR, Rovelink PW, Bruder JT, Wickham TJ, Kovesdi I, Crystal RG. 2004. Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses. J. Virol. 78:2572-2580.

Vincent T, Pettersson RF, Crystal RG, Leopold PL. 2004. Cytokine-mediated downregulation of coxsackie-adenovirus receptor in endothelial cells. J. Virol. 78:8047-58.

Leopold PL, Wendland R, Vincent T, Crystal RG. 2006. Neutralized adenovirus-immune complexes can mediate effective gene transfer via a Fc receptor-dependent infection pathway. J. Virol. 80:10237-10247.

Berth SB, Leopold PL, Morfini G. Virus-induced neuronal dysfunction and degeneration (submitted).

Cystic fibrosis and other pulmonary diseases

Kaner RJ, Worgall S, Leopold PL, Stolze E, Milano E, Hidaka C, Ramalingam R, Hackett NR, Singh R, Bergelson J, Finberg R, Falck-Pedersen E, Crystal RG. 1999. Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor. Am. J. Respir. Cell Mol. Biol. 20:361-370.

Worgall S, Singh R, Leopold PL, Kaner RJ, Hackett NR, Topf N, Moore MAS, Crystal RG. 1999. Selective expansion of alveolar macrophages in vivo by; ex vivo adenovirus-mediated transfer of the murine granulocyte-macrophage colony stimulating factor cDNA to and transplantation of modified macrophages to the lungs of syngeneic mice. Blood 93:655-666.

Harvey BG, Leopold PL, Hackett NR, Grasso T, Williams PM, Tucker A, Kaner R, Ferris B, Gonda I, Ramalingham R, Kovesdi I, Shak S, Crystal RG. 1999. Airway epithelial expression of vector-derived cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts following repetitive endobronchial spray administration of an adenovirus vector expressing the normal CFTR cDNA to individuals with cystic fibrosis. J. Clin. Invest. 104:1245-1255.

De B, Heguy A, Leopold PL, Wasif N, Korst RJ, Hackett NR, Crystal RG. 2004. Intrapleural administration of a serotype 5 adeno-associated virus coding for α1-antitrypsin mediates persistent, high lung and serum levels of α1-antitrypsin. Molec Ther 10:1003-1010.

De B, Heguy A, Hackett NR, Ferris B, Leopold PL, Lee J, Pierre L, Gao G, Wilson JM, Crystal RG. 2006. High levels of persistent expression of α1-antitrypsin mediated by the nonhuman primate serotype rh.10 adeno-associated virus despite preexisting immunity to common human adeno-associated viruses. Molec Ther. 13:67-76.

Heguy A, Harvey BG, Leopold PL, Dolgalev I, Raman T, Crystal R. 2007. Responses of the human airway epithelium transcriptome to in vivo injury. Physiol. Genom. 29:139-148.

Hubner R-H, Kiuru M, Krause A, Leopold PL, Crystal RG. 2009.Dysfunction in glycogen storage with metabolic consequences in a mouse model of Alpha 1-Antitrypsin Deficiency. Am. J. Resp. Cell Molec. Biol. 40:239-247.

Saitoh H, Leopold PL, Harvey BG, O’Connor TP, Worgall W, Hackett NR, Crystal RG. 2009. Emphysema mediated by lung overexpression of ADAM10. Clin Transl Sci (in press).

Song Y, Lou HH, Boyer JL, Limberis MP, Vandenberghe LH, Hackett NR, Leopold PL, Wilson JM, Crystal RG. (2009). "Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing", Hum Gene Ther, (in press)

Regenerative medicine

Sato N, Leopold PL, Crystal RG. 1999. Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog J. Clin. Invest. 104:855-864.

Sato N, Leopold PL, Crystal RG. 2001. Effect of adenovirus-mediated expression of Sonic hedgehog gene on hair regrowth in mice with chemotherapy-induced alopecia. J Natl Cancer Inst. 93(24):1858-1864.

Bergstein, I, Leopold PL, Sato N, Panteleyev AA, Christiano AJ, Crystal RG. 2002. In vivo enhanced expression of Patched dampens the Sonic hedgehog pathway. Molec. Ther. 6:258-64.

Lou H, Crystal RG, Leopold PL. 2005. Enhanced in vivo efficacy of cholesterol-minus Sonic hedgehog. Molec Ther. 12:575-8.

Strulovici Y, Leopold PL, O’Connor TP, Pergolizzi RP, Crystal RG. 2007. Human embryonic stem cells and gene therapy. Molec. Ther. 15:850-866.

Cancer

Hirschowitz EA, Leonard S, Song W, Ferris B, Leopold PL, Lewis J, Bowne WB, Wang S, Houghton AN, Crystal RG. 1998. Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma. Gene Ther. 5:975-983.

Sato N, Leopold PL, Crystal RG. 2001. Effect of adenovirus-mediated expression of Sonic hedgehog gene on hair regrowth in mice with chemotherapy-induced alopecia. J Natl Cancer Inst. 93(24):1858-1864.

Carolan BJ, Heguy A, Harvey BG, Leopold PL, Ferris B, Crystal RG. 2006. Upregulation of expression of the ubiquitin carboxyl terminal hydrolase L1 gene in human airway epithelium of cigarette smokers. Cancer Res 66:10729-40.

Harvey BG, Heguy A, Leopold PL, Carolan B, Ferris B, Crystal RG. 2007. Modification of gene expression of the small airway epithelium in response to cigarette smoking. J. Molec. Med 85:39-53.

Lysosomal storage diseases

Sondhi D, Peterson DA, Giannaris EL, Sanders CT, Mendez BS, De B, Rostkowski A, Blanchard B, Bjugstad K, Sladek JR Jr., Redmond ED Jr., Leopold PL, Kaminsky SM, Hackett NR, Crystal RG. 2005. AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in TPP-1 expression at levels compatible with therapy for late infantile neuronal ceroid lipofuscinosis. Gene Ther 12:1618-32.