Croyle, Maria A., Ph.D.
Associate Professor
PHR 4.214D
512-471-1972
macroyle@mail.utexas.edu

Research Interests

Graphic Depiction of How PEGylation Coats Biomolecules and Protects them from Antibody Neutralization

PEGylation Does Not Compromise Transduction Efficiency of HIV-VSV-G In Vivo. Histological sections from eyes treated with native virus (VSVG) show gene expression is located predominantly in the outer nuclear layer (onl) while those from eyes treated with PEGylated virus (PEG-VSVG) are located in the retinal pigment epithelium (rpe), the outer nuclear later (onl) and the inner nuclear layer (inl) when compared to sections from animals dosed with saline (Ctrl) (magnification 200x). From Croyle et al. J.Virol. (2004)

PEGylation improves the amount of active virus in the circulation and reduces clearance from the systemic circulation. From Wonganan et al. (2009) in review.

PEGylated Adenoviral Vectors Efficiently Transduce Cells That Do Not Express
the Coxsackievirus and Adenovirus Receptor (CAR). A) Comparison of transduction efficiency of unmodified adenovirus (Native) and PEGylated virus on cells that do not express CAR. B) Micrograph of CAR (-) cells treated with adenovirus labeled with Cy3 maelmide fluorescent dye. Very few virions (red) were located in the cytoplasm in contrast to DAPI-stained nuclei (blue). C) Micrograph of CAR (-) cells treated with Cy3 labeled virus conjugated to TMPEG. Cells are loaded with virus (red) in contrast to DAPI-stained nuclei. D) Micrograph of cells treated with free Cy3 dye in PBS (control).

PEGylation does not compromise the transduction efficiency of helper-dependent adenovirus and allows for significant levels of gene expression in immunized mice. Mice given 1 x 1011 particles of adenovirus and euthanized 7 days after administration (or rechallenge) for analysis of beta-galactosidase expression. Similar levels of transgene expression were found in livers of naïve animals treated with unmodified (HD) or PEGylated virus (PEG-HD). Significant gene expression could only be detected in animals rechallenged with PEGylated virus (HD/PEG-HD) after prior immunization with unmodified virus while the gene could not be detected in mice immunized and rechallenged with unmodified virus (HD/HD).
Panel A) Tissue section from a mouse given saline (vehicle control). B) Tissue section from a naïve mouse given unmodified virus. C) Section from a naïve mouse given PEGylated virus. D) Section from a mouse immunized with adenovirus and rechallenged with unmodified virus. E) Section from mouse immunized with unmodified virus and rechallenged with PEGylated virus. From Croyle et al Gene Ther. (2005).

Cytokine Profiles of PEGylated and Non-PEGylated Helper-Dependent Adenovirus

PEGylation Significantly Reduces CTL Production after Administration of PEGylated Ad in the Lung (A) and the Liver (B) From Croyle et al. Hum. Gene Ther. (2002) and Croyle et al. J. Virol. (2001).

Virus Release Profiles for PLGA Microspheres Prepared with Different Organic Solvents Over Time
From Jin Huk Choi, Pharmaceutics Graduate Student

Virus Release Profiles of PLGA Formulations Stored at Various Temperatures for 1, 7 and 30 days.
From Jin Huk Choi, Pharmaceutics Graduate Student

Biochemical Modification of Viruses to Evade the Immune Response

Recombinant viruses were originally thought to be perfect 'Trojan horses' for gene therapy due to their inherent ability to efficiently transduce cellular targets to support their own replication.  Their use is limited, however, by the fact that they are rapidly cleared from the circulation by the immune system preventing them from reaching their target and reducing transduction efficiency if a second dose is needed.  This fostered a decade of research devoted to genetic and molecular manipulation of the virus genome and capsid proteins. Animals treated with these modified viruses, however, still experience acute toxicity initiated by the innate immune response against capsid proteins.

PEGylation, a method for the modification of virus proteins by covalent attachment of polyethylene glycol (PEG) and originally pioneered to reduce the immune response generated against therapeutic proteins, does not significantly compromise virus activity and extends the half-life of the virus in the systemic circulation.  This method also significantly reduces inflammatory cytokines and cytotoxic T cells during the innate immune response and protects vectors from inactivation by complement, neutralizing antibodies and degradation in the gastrointestinal tract.  Current research in the laboratory focuses on characterization of viruses modified by polymers with properties similar to PEG.  Other work in this area focuses on embedding recombinant viruses in complex polymer matricies to protect the virus from harsh physiological environments and the immune system as well as maintain continuous delivery of virus over extended periods of time without developing tolerance to the vector or the transgene product. 

Project Funding: Past and Present

American Association of Colleges of Pharmacy/Burroughs Wellcome Fund

Southwest National Primate Research Center (SNPRC) Pilot Project Grant

UT Undergraduate Research Fellowships

Recent Publications

Wonganan, P., Clemmens, C., Leggiero, E., Brasky, K., Dekker, J., Astone, D., Pastore, L. and Croyle, M.A. Pharmacology and Toxicology of PEGylated Helper-Dependent Adenovirus in Non-Human Primates. 2009 (in review).

Le, H. T., Yu, Q. C., Wilson, J. M. and Croyle, M. A. Utility of PEGylated Recombinant Adeno-Associated Viruses for Gene Transfer.  J. Control. Rel. 2005 108(1):161-177.

Croyle, M. A., Le, H., Linse, K., Ming, X., Toietta, G., Beaudet, A. and Pastore, L. PEGylated Helper-Dependent Adenoviral Vectors: Highly Efficient Vectors with an Enhanced Safety Profile. Gene Ther. 2005 12(7):579-588.

Croyle, M. A. Callahan, S. M., Auricchio, A., Schumer, G., Wilson, J. M., Linse, K., Brunner, L. J. and Kobinger, G. P. PEGylation of a Vesicular Stomatitis Virus G Pseudotyped Lentiviral Vector Prevents Inactivation in Serum. J. Virol.  2004 78(2):912-921.

Cheng, X., Ming, X. and Croyle, M. A. PEGylated Adenoviruses Enhance Gene Delivery to the Intestinal Epithelium by the Oral Route. Pharm. Res. 2003 20(9):1444-1451.

Croyle, M. A., Chirmule, N., Zhang, Y. and James M. Wilson. PEGylation of E-1 Deleted Adenovirus Vectors Allows Significant Gene Expression upon Re-administration to Liver. Hum. Gene Ther. 2002 13(15):1887-1900.

Croyle, M. A., Chirmule, N., Zhang, Y. and Wilson, J. M. "Stealth" Adenoviruses Blunt Cell Mediated and Humoral Immune Responses Against the Virus and Allow for Significant Gene Expression upon Re-administration for Gene Therapy to the Lung. J. Virol. (2001) 75(10):4792-4801.

Croyle, M. A., Yu, Q. C. and Wilson, J. M. Development of a Rapid Method for the PEGylation of Adenoviruses with Enhanced Transduction and Improved Stability Under Harsh Storage Conditions. Human Gene Therapy. (2000) 11:1713-1722.

Recent Presentations at National and International Meetings

BioKorea 2007 International Biotechnology Conference.  September 12-14, 2007. Maria A. Croyle, invited speaker. Talk title: "Pharmacology and Toxicology of Modified Recombinant Viruses for Vaccine and Gene Transfer".

Tenth Annual Meeting American Association Of Gene Therapy, Seattle, W.A.  May 30-June 3, 2006.
Wonganan, P., Leggiero, E., Brasky, K., Dekker, J., Astone, D., Pastore, L. and Croyle, M.A. Toxicology of PEGylated Helper-Dependent Adenovirus in Non-Human Primates.

Fifteenth Annual Meeting of the American Association of Pharmaceutical Scientists.  San Antonio, TX.  October 29 to November 2, 2006.
Boquet, M. P. and Croyle, M. A. Optimization of Ploy(D-L-lactide-co-glycolide) Microsphere Production for Oral Delivery to Promote Adenovirus Stability and Intestinal Gene Transfer

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