Gordon J. Lutz, Ph.D.

Gordon J. Lutz, Ph.D.
Associate Professor

Ph.D. (1994) University of Pennsylvania
Phone: 215-762-2396
Email: glutz@drexel.edu

Nanopolymer-Oligonucleotide Drug Discovery:
Molecular Therapy of Muscular Dystrophy and Other Diseases

Research Interests

Development of novel polymer-oligonucleotide drugs for treatment of Duchenne Muscular Dystrophy
Molecular and mechanical structure-function studies of myosin in skeletal muscle
Cellular and molecular mechanisms of eccentric contraction-induced injury in skeletal muscle
Drug development for cancer therapy using novel nanopolymer-oligonucleotide compounds

Research Projects

I. Development of Novel Polymer-Oligonucleotide Drugs for Treatment of Duchenne Muscular Dystrophy

A major research emphasis of our group is to develop effective novel nanopolymer-oligonucleotide drugs for treatment of Duchenne Muscular Dystrophy (DMD). DMD is the most common fatal heritable disease world-wide, afflicting 1 of 3500 newborn males. DMD is caused by mutations in the gene encoding dystrophin, a critical membrane-associated cytoskeletal protein. The majority of DMD mutations encode early termination signals, or deletions and insertions that produce "out of frame" mRNA transcripts. The loss of functional dystrophin results in progressive muscle weakness throughout the body. Antisense oligonucleotides (AOs) with 2-O-methyl substitutions (2OMeAOs) function by modulating dystrophin pre-mRNA splicing, and are broadly regarded as promising drugs for potential treatment of DMD. Specifically, 2OMeAOs have been shown to facilitate targeted "skipping" of dystrophin exons, enabling the removal of early termination codons or restoration of the correct reading frame, thereby restoring production of a functional protein. However, the main obstacle that has limited the usefulness of 2OMeAOs in treatment of DMD is a lack of adequate carriers to facilitate their delivery to myonuclei.

Recently our group has synthesized a family of cationic nanopolymers comprised of poly(ethylene-glycol) (PEG)-poly(ethylene imine) (PEI), that represent a flexible AO delivery system with controllable size and surface charge, adjustable unpackaging properties, and flexibility for addition of moieties that target specific entities on cell membranes (Figure 1).

Figure 1. Schematic representations of various PEI-PEI-AO polyplexes. PEI amine groups (blue) are connected through ethylene groups (black); attached PEG chains (red) exhibit a random coil conformation. Complexed AO (green) is embedded into PEI or interconnects several copolymers. Individual polyplexes within the aggregate particles are indicated by yellow circles.

We also recently completed a study demonstrating that PEG-PEI copolymers facilitated efficient cellular uptake and nuclear delivery of AO in mature skeletal muscle fibers isolated from mdx mice. Confocal analysis of dual fluorescently tagged PEG-PEI-AO polyplexes at 24 h after transfection, showed the copolymer and AO were co-localized within punctate membrane-associated structures (Figure 2). Importantly, AO was efficiently translocated into myonuclei, while the copolymer was mainly excluded. The morphology of all transfected myofibers was perfectly maintained with no indication of damage or cytotoxicty. Quantitative fluorescence analysis showed transfection with PEG-PEI-AO resulted in a 6-fold higher uptake of AO into myonuclei compared with transfections of AO alone. Overall, our study provides evidence that PEG-PEI copolymers function as high-capacity, non-toxic carriers for efficient delivery of AO to nuclei of mature myofibers.

Figure 2. Confocal images of a representative mdx FDB skeletal muscle fiber at 24 h after transfection with polyplexes comprised of 6-FAM-AO (135 µM) complexed with the rhodamine-labeled copolymer PEI25000(PEG5000)10. The confocal images are from a single optical slice, taken at the approximate mid-plane of the muscle fiber (paraformalehyde fixed), and are shown at three different magnifications. The confocal images (simultaneous 4-channel recordings) included (A-A'') 6-FAM-AO (green), (B-B'') rhodamine-labeled copolymer (red), (C-C'') Hoechst 33342 dye-labeled nuclei (blue), and differential interface contrast (DIC). Panel C-C" shows an overlay of the DIC and Hoechst images, while panel D-D'' shows a triple overlay of the rhodamine, 6-FAM and DIC images. The low and mid magnifications clearly show the localization of AO within all myonuclei in the field of view. The mid and high magnification views show that AO is concentrated within the nuclei (i.e., arrow in A'-A''), while the copolymer is almost completely excluded from entering the nuclei. The red-green-DIC overlay demonstrates that copolymer and AO are co-localized within punctate membrane-associated structures (yellow structures), that are especially abundant in perinuclear regions of the myofiber.

We have also examined the capacity of various PEG-PEI copolymers to enhance AO transfection efficiency and facilitate dystrophin expression in skeletal muscle of mdx mice. Single intramuscular injections of AO complexed with low Mw PEG-PEI copolymers into TA muscles of mdx mice resulted in widespread distribution of dystrophin-positive fibers at 3 weeks after injection, with no evidence of cytotoxicity (Figure 3). We conclude that low Mw PEG-PEI copolymers function as high-capacity, non-toxic AO carriers suitable for in vivo transfection of skeletal muscle, and are promising compounds for potential use in non-viral molecular therapy of DMD.

Figure 3. Dystrophin expression in TA muscles of mdx mice at 3 weeks after a single intramuscular injection of 20 µg AO complexed with a low Mw PEG-PEI copolymer. (A) Dystrophin immunolabeling of a whole TA transverse section obtained from the midpoint along the length of the muscle. The composite image was constructed from individual overlapping images. Dystrophin-positive fibers were broadly distributed throughout the muscle cross-section.

II. Molecular and Mechanical Structure-Function Studies of Myosin in Skeletal Muscle

Myosin is the molecular motor in skeletal muscle. All vertebrates express multigene families of the myosin subunits, myosin heavy chain (MHC) and myosin light chain (MLC) in their skeletal muscle. Together, the isoforms of MHC and MLC are critical determinants of contractile performance of skeletal muscle cells. A major limitation to previous studies is that structure-function analysis of myosin has not been performed in intact “living” muscle cells. We have developed a unique experimental model to study myosin isoform structure and function in single intact cells. Currently these studies are focused on structure-function analysis of MLC. Frog is chosen for these studies because isolated intact frog fibers are a superb preparation for high-resolution measurements of myosin cross-bridge kinetics. In vivo gene transfer methods are used to express high levels of recombinant MLC in frog muscle fibers. Regenerating muscles are co-injected with plasmid expression vectors encoding full-length MLC1 and green fluorescent protein (GFP) and then harvested 3 weeks after plasmid injection. Figure 4 shows the high level of in vivo transfection efficiency that can be achieved with this method. Trans-MLC1 in individual GFP-positive fibers was measured with western blots and found to account for nearly 50% of the total MLC1 (not shown). Confocal microscopy confirmed correct spatial distribution and incorporation of trans-MLC1 within the sarcomeres (not shown). We have performed preliminary single fiber contractile experiments on regenerated fibers that were co-transfected with recombinant MLC1 and CMV-GFP and harvested 3 weeks after plasmid injection. The regenerated-transfected fibers appear to have contractile properties very similar to normal fibers. Currently we are using this model to determine the relationship between the amount of trans-MLC1 and differences in contractile function. The MLC expression vectors will ultimately be engineered to contain modifications at strategic locations thought to be important in regulating myosin cross-bridge kinetics.

Figure 4. In vivo gene transfer of plasmid expression vectors resulted in high transfection efficiency and persistent expression. Numerous transfected (GFP-positive) fibers are observed in the longitudinal (left) and transverse (right) images of the anterior tibialis muscle, harvested 3 weeks after in vivo transfection.

III. Cellular and Molecular Mechanisms of Muscle Injury
When skeletal muscle is forcibly lengthened while activated (eccentric contraction), injury occurs to the muscle that is characterized by a rapid and prolonged loss in force-generating ability followed by delayed onset muscular soreness. Injury to skeletal muscle from eccentric contractions is an extremely common clinical condition that occurs as a result of vigorous exercise and other forms of normal and accidental muscle overuse. Although recent studies have provided important insights into the cellular, biochemical and structural alterations that follow eccentric contraction-induced injury, the precise mechanical conditions, and cellular or molecular mechanisms that cause the injury remain poorly understood. A focus of our laboratory is to characterize mechanical-based muscle injury during eccentric contractions using isolated frog single fibers. The mechanics experiments are performed while monitoring length changes of short (1 mm) segments and sarcomere length transients along the full length of the fiber (Figure 5). This system provides a very precise correlation between mechanical events and muscle injury in intact muscle cells. By coupling this experimental model with in vivo gene transfer and molecular imaging we are exploring the causes of muscle injury at the cellular and molecular levels. In the future we will expand this model to study the remodeling and gene regulation of muscle in response to eccentric contraction-induced injury.

Figure 5. Segment length recordings during eccentric contractions. (Top Left) Single intact muscle fiber mounted between a force transducer and servo motor with surface markers placed at ~ 1mm increments along its length. (Bottom Left) Single frame recording of distance between surface markers (purple-top trace) imaged with a photodiode array . Also shown are force (blue), length (red) and stimulus period (green) as a function of time during a single eccentric contraction. (Right) Length transients of all segments of a fiber recorded simultaneously during a contraction, and resulting force production. The 1st and 10th contraction of an EC protocol are shown. Clearly, following 10 ECs, the fiber has become disorganized, with a high degree of non-uniformity in mechanical output along its length. Force production has dropped significantly following the 10 EC protocol.

IV. Drug Development for Cancer Therapy Using Novel Nanopolymer-Oligonucleotide Compounds

A new class of multilayered drugs that combine chemically-modified antisense oligonucleotides (AOs) with functionalized synthetic polymers are potentially powerful anti-cancer agents. The attractiveness of these compounds stems from the fact that they can be engineered to be highly specific, potent, flexible (adaptable), and long lasting, along with being inherently safe. This project utilizes an integrative strategy to manufacture, structurally analyze, and determine the biological efficacy of a set of novel synthetic nanopolymers specifically designed to deliver AOs to prostate cancer cells in vitro and prostate secondary tumors in vivo. This is a collaborative research effort involving Alessandro Fatatis in our department and Margaret Wheatley in Drexel's School of Biomedical Engineering. The research plan represents a comprehensive Drug Discovery Project that capitalizes on the natural interface between Drexel’s resources in biochemical engineering and pharmacological/physiological biomedical research.

Selected Publications

Williams J. H., Sirsi, S. R., Latta, D., and G. J. Lutz. (2005) Rescue of dystrophin expression by exon skipping in mdx mice following intramuscular injection of antisense oligonucleotides complexed with PEG-PEI copolymers. Molecular Therapy (Submitted: In Revision - pdf available soon).

Glodde, M., Sirsi S.R., and G. J. Lutz. (2005) Physiochemical Properties of Low and High Molecular Weight PEG-Grafted Poly(ethylene imine) Copolymers and their Complexes with Oligonucleotides. Biomacromolecules (ACCEPTED - pdf available soon).

Sirsi, S. R., Williams J. H., and G. J. Lutz. (2005) Poly(ethylene imine)-Polyethylene Glycol Copolymers Facilitate Efficient Delivery of Antisense Oligonucleotides to Nuclei of Mature Muscle Cells of mdx Mice. Hum. Gene Therapy. 16:1307-1317.

Robinson, D. A., Bremner, S. N., Sethi, K., Shah, S.B., Sirsi S.R. and G. J. Lutz. (2005) In Vivo Expression of Myosin Essential Light Chain Using Plasmid Expression Vectors in Regenerating Frog Skeletal Muscle. Gene Therapy. 12(4): 347-357.

Lutz, G. J., Sirsi, S. R., Shapard-Palmer, S. A., Bremner S. N. and R. L. Lieber. (2002) Influence of myosin heavy chain and myosin light chain isoforms on contractile properties of intact muscle fibers from Rana pipiens. Am. J. Physiol. (Cell Physiol.) 282: C835-C844.

Lutz, G. J., Bremner, S. N., Bade M. J. and R. L. Lieber. (2001) Identification of myosin light chains in Rana pipiens skeletal muscle and their expression patterns along single fibers. J. Exp. Biol. 204(24): 4237-4248.

Lutz, G. J., Rizzaghi, S., Cuizon, D. B. and R. L. Lieber. (2000) Cloning and characterization of the S1 domain of four myosin isoforms from functionally divergent fibre types in adult Rana pipiens skeletal muscle. Gene. 250: 97-107.

Lutz, G. J., Cuizon, D. B., Ryan, A. F. and R. L. Lieber. (1998) Four novel myosin heavy chain clones define a molecular basis for fiber types in Rana pipiens. J. Physiol. 508.3: 667-680.

Lutz, G. J., and L.C. Rome. (1994) Built for jumping; the design of frog muscular system. Science. 263: 370-372.