Selection and Use of Ligands for Receptor-Mediated Gene Delivery to Myogenic Cells

WG Feero1,SLi2, JD Rosenblatt3, N Sirianni4, JE Morgan3, TA Partridge3, L Huang2 and EP Hoffman1,4 Departments of 1Human Genetics, 2Pharmacology, and 4Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 3Muscle Cell Biology Group, MRC Clinical Research Centre, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK

Identification of myogenic cell targeting ligands is a critical observed in regenerating fibers of patients with Duchenne step in the development of synthetic vectors for gene deliv- muscular dystrophy and the ability to direct a large enzyme ery to skeletal muscle. Here we describe the screening of conjugate to the cytoplasm of myotubes. Finally, we show six potential targeting ligands (insulin, insulin-like growth that incorporation of transferrin into an artificial virus con- factor I, iron transferrin, gallium transferrin, alpha-bungaro- sisting of poly-L-lysine-condensed DNA coated with a lipid toxin and carnitine) for their ability to bind dystrophin- shell (LPDII formulation) results in ligand-directed delivery deficient myotubes in vitro. Those ligands showing high of DNA to myogenic cells. This is the first report of gene levels of binding to myotubes were then tested on fully dif- transfer to myogenic cells using a ligand-directed synthetic ferentiated, isolated, viable myofibers. Of the ligands vector. These results suggest that rational design of ligand- tested, transferrin showed the most promise based on high directed, fully synthetic, gene delivery vehicles is a viable levels of binding to myogenic cells, high levels of receptor approach to skeletal muscle vector development.

Keywords: myoblast; myotube; gene transfer; liposome; transferrin

Introduction vivo.6,7 Several reports have been published demonstrating the use of nontargeted cationic liposomes8 or cationic The development of highly efficient methods for wide- liposomes combined with polylysine condensed DNA9 to spread, systemic delivery of genes to skeletal muscle will direct gene delivery to myoblasts and myotubes in cul- be critical to the success of gene-based therapies for pri- ture. These studies demonstrate that myoblasts are mary genetic muscular disorders. For example, pheno- efficiently transfected using cationic liposomes, and that typic correction of Duchenne muscular dystrophy is esti- precondensation of DNA with poly-lysine enhances lipo- mated to require restoration of about 20% of normal fection. However, the overwhelming charge interaction 1,2 dystrophin levels in affected muscles. No currently between positively charged liposome–DNA complexes available vector can acheive this efficiency of gene trans- and negatively charged cell membranes and extracellular fer in a tissue that represents 30% of a patient’s body matrix makes specific targeting difficult. A possible sol- mass. Traditional approaches for gene delivery to skeletal ution to this problem is the use of a newly developed muscle have focused on the intramuscular injection of liposomal vector, LPDII, which can be manipulated to be viral vectors (adenovirus (ADV), retrovirus (RV), herpes neutral or anionic in overall charge and thus able to inter- virus type 1 (HSV-1)). An alternative avenue, largely act with cells in a ligand-dependent manner.10 Such vec- unexplored for skeletal muscle, is to develop rationally tors are similar in size to viral vectors with an average designed, fully synthetic gene delivery vectors for sys- diameter of 100–150 nm, and have been shown to temic delivery. Such vectors could be designed to be of mediate transient transduction of several cell types (L low immunogenicity/toxicity, targeted to skeletal Huang, personnal communication). We hypothesized muscle, stable in the bloodstream, produced easily on a that charge-neutral, entrapped DNA liposomes could be large scale and capable of mediating transduction with developed which would show transduction of myogenic high efficiency. cells in a ligand-dependent manner. Most efforts to develop targeted synthetic vectors have Here we describe the rational design of a synthetic vec- 3,4 taken advantage of receptor-mediated endocytosis to tor for ligand-directed gene delivery to myogenic cells. direct gene delivery to nonmyogenic cells. For example, First, candidate muscle binding ligands were screened to the use of synthetic vectors to deliver genes to hepato- quantify their levels of association with myotubes gener- 5 cytes has yielded promising results both in vitro and in ated from a conditionally immortalized, nontumorigenic, dystrophin-deficient myogenic murine cell line.11 Second, ligands identified as having high levels of association Correspondence: EP Hoffman, W1211 BST, University of Pittsburgh with myotubes were screened for their ability to bind iso- School of Medicine, Pittsburgh, PA 15261, USA lated, viable, myofibers derived from mice as well as for Received 23 November 1996; accepted 24 March 1997 the presence of ligand receptor in muscle of Duchenne Ligands for gene delivery to myogenic cells WG Feero et al 665 muscular dystrophy patients. Third, selected ligands otoxin15 (2.31 ± 0.656 pmoles/mg cell protein). Carnitine16 were tested for their ability to direct a large macromol- (0.831 ± 0.156 pmoles/mg cell protein) and IGF-117,18 ecule (avidin/␤-galactosidase) to myogenic cells in vitro. (0.147 ± 0.056 pmoles/mg cell protein) showed an inter- Finally, one of the identified ligands was tested for its mediate level of association. Specific binding of insulin19 ability to direct transfection of myogenic cells by LPDII. was below the level of detection. Based on these results, iron transferrin and bungarotoxin were selected for Results additional testing. Transferrin but not bungarotoxin shows maturation- Transferrin and alpha-bungarotoxin show high levels of dependent decline in total association with myogenic myotube association cells We applied three criteria to select a subgroup of known Myotubes represent a very early stage in the differen- muscle-binding ligands for biochemical screening. First, tiation of myogenic cells, therefore it is important to the preferred molecular weight of the ligand was less examine the levels of ligand binding to fully differen- than 100 kDa, based on the rationale that the ligand tiated myogenic cells. This was accomplished by study- should not contribute greatly to the size of the gene deliv- ing the binding of bungarotoxin and iron transferrin to ery vehicle into which it was eventually to be incorpor- freshly dissociated, viable, myofibers isolated from mice ated. Second, ligands were chosen that show binding to (Figure 2a and b). These fibers display characteristics of mature skeletal muscle through a cell surface receptor highly differentiated muscle, including complete basal with relatively high affinity (sub-micromolar K ). Finally, d laminae, well-organized neuromuscular junctions, and the ligands selected were amenable to chemical modifi- myotendinous junctions.20–22 cations allowing radiolabeling or attachment to macro- Ligand association with extensor digitorum longus molecules for delivery to myogenic cells. Based on these (EDL) myofibers isolated from 2-month-old mdx mice criteria six ligands were chosen for screening. under conditions identical to those used for ligand bind- Dystrophin gene and protein replacement in vivo is a ing to myotubes (200 nm, 1.5 h, 37°C) revealed much long-term goal of these studies. Because dystrophin lower levels of transferrin than alpha-bungarotoxin deficiency may have unknown effects on receptor levels in myotubes and myofibers, dystrophin-deficient myotubes seemed the most appropriate cell with which to screen for ligand binding.11 Binding studies of radiolabeled ligands at a fixed concentration (200 nm) with dystrophin-deficient myotubes (Figure 1) showed that three of the six had high levels of association (Ͼ1 pmol/mg cell protein): iron transferrin12,13 (1.56 ± 0.417 pmoles/mg cell protein), gallium transferrin14 (1.18 ± 0.263 pmoles/mg cell protein), and alpha-bungar-

Figure 1 Candidate targeting ligands show variable levels of association with dystrophin-deficient myotubes. The specific association of six radiolabeled candidate targeting ligands with dystrophin-deficient myotubes was Figure 2 Isolated viable myofiber preparations for ligand-binding studies. measured in vitro. Under identical binding conditions (200 nm ligand Single fibers were prepared from extensor digitorum longus muscle of a concentration, 1.5 h, 37°C) levels of observed binding in decreasing order mature normal mouse and incubated for 10 min in trypan blue (a). Note were as follows, alpha-bungarotoxin (BUNG) Ͼ iron transferrin (FeTF) that the fiber appears continuous and intact, excluding trypan blue Ͼ gallium transferrin (GaTF) Ͼ carnitine (CARN) Ͼ insulin-like growth throughout its length. In contrast, below and to the left of the intact fiber factor 1 (IGF-1) Ͼ insulin (INS). The lack of detectable association with is a deceased, hypercontracted fiber which is unable to exclude trypan insulin was probably a result of very low levels of receptor (n = 3 or blue. These fibers can be prepared for binding studies with a minimum of greater for each ligand). contaminating nonmuscle tissue (b) (scale bar = 250 ␮m). Ligands for gene delivery to myogenic cells WG Feero et al 666 association (0.025 ± 0.014 pmoles/mg cell protein and 1.430 ± 0.314 pmoles/mg cell protein, respectively) (Figure 3a). The level of alpha-bungarotoxin association was comparable to that measured in myotubes, while the level of transferrin association was approximately 60-fold lower than that with myotubes. Transferrin association with mdx myoblasts was also lower than the association with myotubes (0.843 ± 0.126 pmoles/mg cell protein, data not shown). Transferrin association with normal mouse (C57BL/6J or C57BL/10J) skeletal muscle was determined using single fibers isolated from hind-limb anterior compartment (5-day, 1-month, and 2-month mice) (Figure 3b) and posterior compartment (5-day-old, 2-week-old and 1-month mice) (Figure 3c) muscles. Transferrin association decreased by three-fold for both anterior and posterior compartment muscles between day-5 and 1-month-old animals. These observations suggest that transferrin association per milligram of cell protein peaks in myotubes, then declines with fiber maturation. The abrupt decline in transferrin association between myotubes and single fibers is probably exagger- ated by an increase in contaminating nonfiber protein in the single fiber binding assay. However, the maintenance of high levels of alpha-bungarotoxin association with single fibers suggests that this error is probably not large.

Transferrin receptor is present in elevated levels in regenerating fibers in patients with Duchenne muscular dystrophy Based on the above observation that transferrin association is greatest with intermediately differentiated murine myogenic cells, we hypothesized that elevated levels of transferrin receptor should be present on regenerating human myofibers. To test this hypothesis, immunofluorescence was used to examine transferrin receptor levels in frozen sections of muscle biopsies from normal and Duchenne muscular dystrophy patients (in which regenerating fibers are a prominent histological feature23). Transferrin receptor staining in normal individuals was faint, punctate and almost entirely associated with the sarcolemma (Figure 4a). In Duchenne patients, however, regenerating fibers (small, eosinophilic and centrally nucleated by hematoxylin and eosin staining) showed intense cytoplasmic as well as sarcolemmal staining (Figure 4b). In contrast, transferrin receptor staining in fully mature fibers was not different from that observed in the normal biopsy. Sections to which no primary antibody was added showed no staining (data not shown). These observations suggest transferrin receptor levels are highly elevated in regenerating myofibers in humans. Figure 3 Alpha-bungarotoxin and iron transferrin binding as a function of developmental stage of myogenic cells. The specific association of alpha- Transferrin, but not bungarotoxin, can direct targeted bungarotoxin (BUNG) and iron transferrin (FeTF) with isolated, viable delivery of an enzyme to myogenic cells murine myofibers under conditions identical to those used for association m ° To test the ability of transferrin and alpha-bungarotoxin studies with myotubes (200 n ligand, 1.5 h, 37 C) was measured. The level of bungarotoxin association to mature myofibers (mf) from EDL mus- to deliver macromolecules to myogenic cells, we bound cle of 2-month-old mdx mice shows a 30% decrease in binding relative to biotin–transferrin (degree of substitution 1.4) or biotin that observed in dystrophin-deficient myotubes (mt) (a). In contrast, FeTF alpha-bungarotoxin to a commercially available avidin– showed a 60-fold decrease in binding of myofibers compared with myotubes ␤-galactosidase complex. These complexes were then (a, left). FeTF association decreases with single fibers isolated from hind- used to qualitatively and quantitatively determine limb anterior compartment muscle (b, right) and posterior compartment ligand-specific association of ␤-galactosidase with muscle (c) of normal mice of increasing ages. This suggests that FeTF transferrin receptor function is developmentally regulated and that FeTF dystrophin-deficient myotubes in vitro. may not be a suitable targeting ligand for fully mature skeletal muscle (n Biotinylated ligands alone bound myotube receptors = 3 or greater aliquots per time-point). with estimated disassociation constants of less than 100 nm. Furthermore, addition of streptavidin modified Ligands for gene delivery to myogenic cells WG Feero et al 667 activity was not limited to the endosomal or lysosomal compartment. Similar experiments were carried out using biotin–bungarotoxin as the targeting ligand. Despite the observed association of radiolabeled bungarotoxin and biotinylated bungarotoxin with myotubes, no ligand-specific association of ABG with myotubes was observed (data not shown). This suggests that complexing ABG to biotin–alpha-bungarotoxin may result in steric hindrance, blocking the ability of bungarotoxin to bind the acetylcholine receptor on the surface of myotubes. Alternatively, biotin–bungarotoxin may not associate with avidin–␤- galactosidase in an orientation that permits binding of bungarotoxin to the acetylcholine receptor. To quantify biotin–transferrin-driven association of ABG with myotubes and myoblasts and to determine the extent of endocytosis of the complex, cells were incubated at 37°C and 0°C in the presence of targeted ABG or ABG plus free transferrin. Quantitative assay of biotin– transferrin-directed association of ␤-galactosidase activity showed myoblasts and myotubes bind essentially equal amounts of the enzyme conjugate at 0°C (22.7 ± 2.8 pmol/mg cell protein, and 23.9 ± 2.0 pmol/mg cell protein, respectively) (Figure 7). However, at 37°C, the amount of enzyme association with myotubes (59.67 ± 5.65 pmol/mg cell protein) was approximately two-fold that observed in myoblasts (35.8 ± 8.4 pmol/mg cell protein). These observations suggest that the bound ligand–enzyme complex is internalized and that the mechanisms of internalization may change with myogen- esis. Figure 4 Regenerating fibers in the skeletal muscle of Duchenne muscular dystrophy patients show high levels of transferrin receptor. Immunofluo- rescence staining for transferrin receptor (monoclonal antibody B3/25) in Lipid-polylysine-DNA (LPDII) particles with covalently normal human skeletal muscle from a 4-year-old individual (a) and in incorporated transferrin transduce myoblasts and muscle from a 3-year-old patient with Duchenne muscular dystrophy (b) myotubes in a ligand-dependent manner was performed. In normal fibers transferrin receptor staining was weak, To test the ability of transferrin to direct DNA to myo- punctate and nearly all associated with the sarcolemma. In contrast, areas genic cells in culture, we developed a novel targeted arti- of regenerating fibers in dystrophin-deficient muscle show very intense ficial virus modeled after the previously described folate- staining for transferrin receptor both at the sarcolemma and throughout 10 the myofiber cytoplasm. targeted liposome preparation. This artificial virus was constructed by first condensing plasmid DNA containing a reporter gene using poly-l-lysine followed by interac- the binding characteristics of both biotinylated ligands tion of the DNA–polylysine complexes with transferrin- (data not shown). These observations suggested biotin modified, DOPE/CHEMS liposomes. These transferrin- modified transferrin and alpha-bungarotoxin maintain targeted LPDII particles containing DNA encoding ␤- the ability to bind their respective myotube receptors galactosidase or luciferase reporter genes were then used with high affinity. to transfect both myoblasts and myotubes. Myoblasts Biotin–transferrin was bound to an enzyme conjugate were transfected 24 h after plating, then switched to dif- (avidin–␤-galactosidase (ABG)) and the delivery of ferentiation media, and harvested at day 2 and 5 of differ- enzyme to myogenic cells was tested with and without entiation for measurement of luciferase activity, or fixed excess unlabeled transferrin. Cytochemical staining of with 2% glutaraldehyde and stained for ␤-galactosidase myotubes for ␤-galactosidase activity revealed a low activity (day 5 only), as previously described. Myotubes level of nonspecific association of ABG with myotubes were transfected at day 5 of differentiation, and assayed (Figure 5a). Complexing ABG with biotin–transferrin 2 days later. For transfections, cells were incubated for greatly enhanced the level of myotube- 5 h at 37°C with 2 ␮g of DNA complexed in either associated ␤-galactosidase activity (Figure 5b). This com- untargeted LPDII, transferrin-LPDII, transferrin-LPDII plex was soluble, as 15-min microcentrifugation of the plus excess free transferrin (11.3 ␮m) or lipofectamine. targeted enzyme complex before addition to the ␤-Galactosidase staining at day 5 of differentiation myotubes did not detectably alter association (Figure 5c). showed that nontargeted LPDII was extremely inefficient Addition of excess free transferrin greatly reduced the at generating transduced myotubes from transfected amount of cell-associated enzyme activity (Figure 5d), myoblasts (Figure 8a). In contrast, transfection of myo- demonstrating that the observed uptake of biotin–trans- blasts using transferrin-targeted LPDII resulted in ferrin–avidin–␤-galactosidase is not simply a result of approximately 5% transduced myotubes (Figure 8b). This transferrin-stimulated, nonspecific endocytosis. The uni- level of transduction was similar to that observed using form distribution of staining observed in association with the cationic liposome formulation, lipofectamine (data the myotubes (Figure 6) suggests that the ␤-galactosidase not shown). We next tested the ability of transferrin- Ligands for gene delivery to myogenic cells WG Feero et al 668

Figure 5 Biotin–transferrin directs a large enzyme conjugate (avidin–␤-galactosidase) to dystrophin deﬁcient myotubes. Cytochemical staining for ␤- galactosidase activity was performed on myotubes incubated for 3 h at 37°C in the presence of: avidin–␤-galactosidase alone (a); avidin–␤-galactosidase coupled to biotin-transferrin (b); avidin–␤-galactosidase coupled to biotin–transferrin which had been centrifuged before addition to myotube cultures (c); avidin–␤-galactosidase coupled to biotin–transferrin plus excess native transferrin (d). The low level of staining evident in a compared with that seen in b suggests that biotin–transferrin directs a soluble (c) biotin–transferrin–avidin–␤-galactosidase complex to myotubes. The ability to compete away uptake using free transferrin (d) suggests that the incorporated biotin–transferrin directly mediates complex uptake, rather than through stimulation of nonspeciﬁc endocytosis (scale bar = 100 ␮m).

LPDII to transfect fully differentiated (day 5) myotubes. (Figure 8d). The great decrease in transduction observed ␤-Galactosidase staining of myotubes showed that trans- with maturation of myoblasts to myotubes suggests that ferrin-LPDII resulted in only one or two visibly trans- the post-binding fate of transferrin-LPDII differs between duced myotubes per well (data not shown). these cell types. To examine more quantitatively the transduction of both myoblasts and myotubes, transfection experiments Discussion were carried out using luciferase as the reporter gene. Luciferase assay of lysates of myotubes generated from Our long-term goal is to develop a fully synthetic gene myoblasts transfected with untargeted LPDII, transferrin- delivery vehicle for systemic gene therapy of muscle in LPDII, transferrin-LPDII plus excess transferrin and lipo- vivo. In this article we describe intermediate steps fectamine confirmed the observations made using the ␤- towards this goal: we describe experimental systems for galactosidase reporter gene (Figure 8c). Incorporation of screening of potential ligands, identification and testing transferrin into LPDII yielded a 16-fold enhancement of of candidate ligands, and construction of a fully synthetic reporter gene activity over untargeted LPDII, with total targeted liposome which transduces myoblasts at a high levels of transfection slightly better than those achieved efficiency. To select compounds for testing as targeting using the cationic liposome, lipofectamine. Importantly, ligands for skeletal muscle, we considered the amount of free transferrin was able to compete away transfection functional receptors on target cells, the cell specificity of by transferrin-LPDII, while a nonspecific protein (bovine receptors, the ability to modify ligands for coupling to serum albumin) had no effect on transduction (data not delivery vehicles, and the intracellular fate of ligand– shown). This suggests that the transduction of myoblasts receptor complexes. Of these, we chose to use the amount by transferrin-LPDII resulted from transferrin-mediated of functional receptor as the primary selection criterion, uptake of the particle rather than from a nonspecific based on the relatively low transduction efficiency of cur- effect of transferrin on cellular endocytic activity. Ident- rent synthetic vectors in skeletal muscle, the fact that ical trends in transduction efficiency were observed when muscle represents a very large target organ, and that myotubes were transfected, although the levels of muscle-specific gene promotors are available.24 reporter gene expression were approximately 10-fold less The lack of quantitative ligand association data for Ligands for gene delivery to myogenic cells WG Feero et al 669

Figure 6 Biotin–transferrin directs avidin–␤-galactosidase to myotube cytoplasm. Shown is a high power image of a multinucleate myotube stained for ␤-galactosidase activity subsequent to incubation for 3 h at 37°C in the presence of avidin–␤-galactosidase coupled to biotin transferrin. The lack of punctate cytoplasmic staining suggests that enzyme activity is not limited to the endosomal or lysosomal compartment in the myotube cytoplasm (scale bar = 40 ␮m).

and 2.31 ± 0.656 pmoles/mg cell protein, respectively). Ligands showing lower levels of association (gallium transferrin, 1.18 ± 0.263 pmoles/mg cell protein; carnitine, 0.813 ± 0.156 pmoles/mg cell protein; insulin-like growth factor I, 0.147 ± 0.056 pmoles/mg cell protein; insulin, not detectable) were excluded from further development in this study. Future artificial vectors may have higher transduction efficiency which will make receptor quantity less important and hence, ligands with low levels of highly muscle-specific receptors more useful targeting candidates. Our concern that myotubes may not accurately represent the ligand-binding ability of fully mature skeletal muscle in vivo led us to adapt a method for single fiber preparation to ligand association studies. This provided us with an in vitro system in which the levels of ligand association with fully mature, living muscle could be quantified (Figure 3). Furthermore, we have previously Figure 7 Biotin–transferrin coupled to avidin–␤-galactosidase is internal- shown that single fibers can be prepared from murine ized by myotubes and myoblasts. Biotin–transferrin-specific association of models of human genetic diseases (eg dystrophin- avidin–␤-galactosidase with myoblasts (MB) and myotubes (MT) at 0°C ° deficient mice (mdx) and merosin-deficient mice and 37 C was measured by ONPG assay (see Materials and methods). (dy/dy)) and that these can be transduced by viruses.22,25 Myotubes were incubated in the presence of biotin–transferrin coupled to avidin–␤-galactosidase or avidin–␤-galactosidase plus unbiotinylated Thus, single fibers provide a good in vitro model for transferrin, specific association was defined as the difference in ␤-galacto- future studies of skeletal muscle gene delivery via tar- sidase activity between these conditions. Myotubes show a higher degree geted synthetic vectors. of conjugate uptake (as measured by the difference between association at Using the single fiber system, we found that the levels ° ° = 37 C and 0 C) than do myoblasts (n 3 or greater wells per condition). of transferrin association with myogenic cells changed as a function of cell maturation. The greatest association was skeletal muscle in the literature motivated us to screen observed in myogenic cells of intermediate differen- candidate ligands for association with dystrophin- tiation (cultured myotubes (1.56 ± 0.417 pmoles/mg cell deficient myotubes under constant binding conditions. Of protein)), while low levels of association were seen with the six ligands screened, iron transferrin and alpha- fully mature EDL fibers from 2-month-old mice bungarotoxin were selected for further testing as tar- (0.025 ± 0.014 pmoles/mg cell protein). This represents a geting ligands due to their high levels of specific associ- 60-fold decrease in cell association of transferrin with ation with myotubes (1.56 ± 0.417 pmoles/mg cell protein maturation. In contrast, bungarotoxin association with Ligands for gene delivery to myogenic cells WG Feero et al 670

Figure 8 Transferrin directs receptor-mediated gene delivery to myogenic cells using a fully synthetic ‘artificial virus’. Cytochemical staining for ␤- galactosidase activity or measurement of luciferase gene expression was performed on myogenic cells transfected with several liposome-based gene delivery vehicles. Myoblasts were transfected with LPDII, an artificial virus containing 2 ␮gofa␤-galactosidase reporter gene encoding plasmid DNA condensed with poly-lysine DNA entrapped in a lipidic shell, without (a) or with covalently incorporated transferrin (b). Without transferrin, no transduced myotubes were visible, while targeting with covalently incorporated transferrin resulted in approximately 5% transduced myoblasts (scale bar = 100 ␮m). (c and d) The result of luciferase assays on supernatants of transfected myoblasts harvested after 2 and 5 days of differentiation (c) or myotubes harvested 2 days after transfection with a variety of lipid-based gene delivery systems. LPDII, unmodified, anionic lipid encapsulated poly-lysine condensed DNA particle; TF-LPDII, transferrin-modified, anionic lipid encapsulated poly-lysine condensed DNA particle; TF-LPDII + TF, transferrin-modified, anionic lipid encapsulated poly-lysine condensed DNA particle plus excess free transferrin (11.3 ␮m); LFA, cationic, DNA–lipofectamine complex (scale bar = 100 ␮m).

EDL fibers from 2-month-old mice (1.43 ± 0.314 fluorescently tagged molecules and found the uptake to pmoles/mg cell protein) was well preserved, maintaining be segmental on the myotube and that the endocytosed about 60% of the association observed in myotubes. Thus, substance was limited to vesicles in the cytoplasm.26,27 the choice of ligand for receptor-mediated gene delivery Our results confirm the previously observed segmental may depend on the differentiation state of the target cell nature of HRP uptake by myotubes,27 and suggest that and the specific disease involved. The latter point is this phenomenon is associated with both receptor- underscored by our observation that regenerating mediated as well as nonspecific endocytic processes. myofibers in human patients with Duchenne dystrophy Finally, we conclude that transferrin is an appropriate show high levels of transferrin receptor while fully choice for directing proteins to myogenic cells via mature fibers in normal muscle do not (Figure 4). receptor-mediated endocytosis. The success with trans- We have shown that transferrin can deliver a func- ferrin conjugates contrasts with the failure of bungaro- tional, high molecular weight enzyme conjugate (avidin– toxin conjugates to deliver functional ␤-galactosidase to ␤-galactosidase) to myotubes. Thus, the transferrin recep- myotubes despite the observed high levels of radiolab- tor on the surface of myogenic cells is not sterically hin- eled bungarotoxin association. This suggests that biotin– dered and can bind transferrin derivatized with high bungarotoxin was unable to associate with the avidin–␤- molecular weight complexes. In addition, internalization galactosidase conjugate in an orientation which permit- of a transferrin-conjugated enzyme does not result in ted it to interact with the acetylcholine receptor. immediate inactivation of the enzyme. This suggests that We have shown that transferrin-LPDII can mediate the transferrin-targeted enzyme conjugate was internal- transferrin-directed gene delivery to myoblasts as well ized by the myotube in an active form (Figures 5–7). as myotubes. The level of transduction achieved by Others have examined myotube and skeletal muscle transferrin-LPDII was roughly equivalent to that of the uptake of nontargeted horseradish peroxidase (HRP) or cationic liposome lipofectamine (Figure 8c and d), but Ligands for gene delivery to myogenic cells WG Feero et al 671 was almost entirely dependent on the covalent incorpor- does not rely on receptor-mediated endocytosis for ation of transferrin as a targeting ligand, as untargeted efficient transduction.34 It is unclear if the same LPDII showed no transduction of myoblasts by ␤-galacto- mechanism(s) governing transduction efficiencies in vitro sidase staining (Figure 8a). This result demonstrates the are also responsible for the maturationally dependent feasibility of developing fully synthetic vectors for recep- decrease in efficiency of viral vector-mediated gene deliv- tor-mediated gene delivery for muscle. Previously we ery observed in vivo.22,35 Future studies will be directed at have shown the ability to achieve targeted delivery of identifying and circumventing maturationally occurring LPDII to cancer cells using folate, a small (441 Da) impediments to efficient transduction of mature muscle. enzyme cofactor. This current work extends our previous data by showing that LPDII can be targeted by a large Materials and methods (80 000 Da) protein, transferrin. A critical point is that the efficiency of transduction of myogenic cells using trans- Preparation of radiolabeled ligands ferrin-LPDII must be enhanced before moving into in vivo Radiolabeled ligands were purchased or prepared using studies. This is illustrated by our observations that by Iodogen (Pierce, Rockford, IL, USA) as previously 36 125 transfecting myoblasts with transferrin-LPDII under opti- described. Saturation of I human apo-transferrin mal conditions we can generate only about 5% trans- (Sigma, St Louis, MO, USA) with five- to 10-fold molar +++ +++ duced myotubes, while with HSV-1 and ADV vectors we excess of iron (Fe ) or gallium (Ga ) was confirmed 37 can achieve essentially 100% transduction. Futhermore, spectrophotometrically with parallel studies of unlab- preliminary in vitro studies on single isolated myofibers eled apotransferrin. It is thought that mammalian trans- and in vivo studies in newborn animals suggest trans- ferrins are interchangeable with regard to receptor speci- 38 ferrin-LPDII has very low ability to transduce myofibers ficity, therefore human transferrin was used in place of (unpublished observations). Enhancement of transduc- mouse transferrin in all binding studies. Specific activities tion efficiency might be accomplished by further enabling of ligands as tested ranged from 5–81 Ci/mmol. transferrin-LPDII to fuse with cells through incorporation 28 Myogenic cell preparation and ligand association studies of fusigenic peptides, by altering the choice of targeting Conditionally immortalized myoblasts derived from ligand, by enhancing the nuclear transport of the particle, male mice hemizygous for mdx and heterozygous for H- or by lessening the dependence on the nuclear transcrip- b 11 ° 29 2K -tsA58 were grown at 34 C in growth media con- tional apparatus. sisting of DMEM supplemented with 10% horse serum, Our data showed that myotubes were 10-fold less 20% fetal calf serum, 2% l-glutamine, 1% penicillin- efficiently transduced than myoblasts by transferrin- streptomycin, 1% chick embryo extract and 20 U/ml of LPDII (Figure 8c and d). Two lines of evidence suggest recombinant murine gamma interferon (Gibco-BRL, that the decrease in transfection efficiency with myogenic Bethesda, MD, USA) (permissive conditions). For gener- differentiation was not simply a result of decreased ation of myotubes, myoblasts were plated at high density numbers of transferrin receptors on the post-mitotic (3 × 105 per 35 mm dish, 6 × 104 per well on 24 well plates, myotubes. First, myotubes bound nearly two-fold more or 1 × 104 per well on 96-well plates) on Matrigel 125I transferrin than myoblasts (1.56 ± 0.417 pmoles/mg ± (Collaborative Biomedical Products, Bedford, MA, USA) cell protein and 0.843 0.126 pmoles/mg cell, coated plates (0.1 mg/ml) in differentiation media respectively), which is in agreement with the results of (DMEM containing 1.25% horse serum, 1.25% fetal calf others that have shown myoblasts have fewer transferrin l 30,31 serum, 2% -glutamine, and 1% penicillin-streptomycin) receptors than do myotubes. Second, myotubes and grown at 37°C (non-permissive conditions). showed approximately two-fold greater levels of associ- Myotubes, some of which displayed spontaneous con- ation and internalization of transferrin-targeted avidin– tractions, were used at day 4–6 after switch to differen- ␤-galactosidase than did myoblasts (59.67 ± 5.65 ± tiation media. Myoblasts were used the day after plating pmol/mg cell protein and 35.8 8.4 pmol/mg cell pro- in growth media without gamma interferon at 37°C (non- tein, respectively). It seems likely, therefore, that the dif- permissive conditions). Protein determinations were car- ference in transduction efficiency is the result of a change ried out using the DC protein assay (Bio-Rad, Hercules, in how myotubes handle the transferrin-LPDII particle CA, USA). Protein contributed to cell lysates by the after binding. Interestingly, others have observed a Matrigel coating of plates was below limits of detection similar, promotor-independent decrease in transfection of the assay. efficiency between myoblasts and myotubes with untar- 8,9 32,33 Association of ligands with dystrophin-deficient geted cationic liposomes and adenoviral vectors. myotubes at 5–7 days of differentiation or myoblasts the Together these observations suggest that there may be a day after plating was determined as follows. Cells were common basis for the decreased transduction efficiency pre-incubated at 37°C for 1.5 h in DMEM containing 2.5% of myotubes by these gene delivery vehicles. Possible bovine serum albumin. Radiolabeled ligands were then mechanisms for such a decrease in transduction added to a final concentration of 200 nm and allowed to efficiency include: decreased endocytosis of large incubate with cells for 1.5 h at 37°C. Nonspecific binding complexes (although our data showing high levels of was measured in the presence of at least 25-fold excess transferrin-directed enzyme conjugate uptake by unlabeled ligand. Cells were rinsed four times with phos- myotubes argues against this); a decreased ability of phate-buffered saline (PBS), lysed in 0.1 m NaOH and endocytosed particles to gain access to the cytoplasm of radioactive incorporation determined using a gamma or myotubes; increased degradation of internalized DNA; liquid scintillation counter. decreased ability of DNA to reach the nuclear transcrip- tional apparatus; and/or altered handling of exogenous Single fiber association studies DNA at the level of the nucleus. Importantly, HSV-1 Normal (C57BL/10J or C57BL/6J) and mdx (dystrophin- infects myoblasts and myotubes equally well in vitro, and deficient, C57BL/10ScSn mdx) mice were bred in insti- Ligands for gene delivery to myogenic cells WG Feero et al 672 tutional animal facilities or obtained from Jackson Lab- ligands in subsaturating concentrations (1.2 nm for trans- oratory (Bar Harbor, ME, USA). Immediately after the ferrin, 1 nm for alpha-bungarotoxin) were incubated with death of the mice, single fibers were prepared from dis- cells for 1.5 h at 0°C in the presence of increasing sected soleus or extensor digitorum longus (EDL) muscle amounts of biotinylated ligand or biotinylated ligand by enzymatic disaggregation in 0.2% type 1 collagenase with constant (at least a four-fold molar excess) of strep- (Sigma) as described elsewhere.39 Due to the small size tavidin. Cells were rinsed three times in PBS, lysed in of the animals less than 5 days old, anterior compartment 0.1 m NaOH, and radioactive incorporation determined and posterior compartment muscles were removed en using a gamma counter. bloc from the lower leg for disaggregation. For 2-week or To determine qualitatively if biotin-modified ligands older animals, the EDL muscle was removed as a rep- were capable of directing avidin–␤-galactosidase to cells, resentative anterior compartment muscle, while the myotubes prepared as described above were incubated soleus muscle was removed from the posterior compart- at 37°C for 3 h in serum-free media containing either ment. Fibers were purified from smaller contaminants avidin–␤-galactosidase (ABG, mw 223 200–322 200 Da (small pieces of connective tissue, free cells) by rinsing depending on lot; Sigma), biotinylated ligand plus ABG the fibers in DMEM, then decanting the DMEM from (combined 15 min before addition to the cells at a 1:1 above the fibers immediately after fiber settling. Fiber molar stoichiometry) or biotinylated ligand plus ABG viability was determined using trypan blue exclusion; in plus a Ͼ25-fold molar excess of free unmodified ligand. practice there was a near 100% correlation between fiber Final biotinylated ligand concentrations were Ͼ75 nm. death and visible hypercontraction. Cells were rinsed once with DMEM/2.5% BSA and three Three aliquots of pooled fibers from four to six muscles times with PBS, fixed in 2% glutaraldehyde (Sigma) and in a small amount of medium (100–200 ␮l) were incu- stained for ␤-galactosidase activity.40 bated at 37°C for 1.5 h in the presence of 200 nm radiolab- To assess quantitatively the ability of biotin–transferrin eled ligand; nonspecific binding was measured in at least to direct ABG to myoblasts and myotubes, cells prepared 25-fold excess unlabeled ligand. After rinsing in DMEM as above were incubated with biotin–transferrin com- (three times) and PBS (once), fibers were lysed in 0.1 m plexed ABG (combined 15 min before addition to cells at NaOH and radioactive incorporation determined using a a 1:1 molar ratio of biotin–transferrin to avidin in the gamma counter. Protein was determined as for ABG complex, final concentration in biotin–transferrin, myotubes. 700 nm) or free native transferrin plus ABG similarly prepared. Cells were incubated for 3 h at 0°Cor37°C, rinsed Immunofluorescence for transferrin receptor as described above, then collected by brief (3 min) tryp- Flash frozen muscle biopsies from two normal (age 4 sinization (1 × trypsin/EDTA (Sigma)). Rinsed cell pel- years) and two individuals with Duchenne muscular dys- lets were resuspended in 100 ␮l of 0.25 m Tris (pH 7.8) trophy (age 3 years) were processed for transferrin then disrupted by three freeze–thaw cycles followed by immunofluorescence. Briefly, unfixed, 8 ␮m cryosections 30 s of sonication (model w380; HeatSystems-Ultrasonics, were thawed on Superfrost Plus (Fisher, Pittsburgh, PA, Farmingdale, NY, USA). ␤-Galactosidase activities were USA) slides, and incubated for 2 h at room temperature determined by o-nitrophenyl-␤-d-galactopyranoside in phosphate-buffered saline/10% horse serum (PBS/HS) (ONPG) assay,41 and cell protein was determined as containing monoclonal antibody against human trans- above. Specific association was defined as biotin– ferrin receptor (1:40, MAB clone B3/25 (Boehringer- transferrin/ABG association minus transferrin/ABG Mannheim, Indianapolis, IN, USA)). For each biopsy, association. parallel sections without primary antibodies were also processed. After rinsing with PBS, biotinylated sheep Transferrin-lipid-polylysine-DNA (TFLPDII) constructs, anti-mouse antibody was applied (1:250 (Amersham, lipofection, and reporter gene assays Arlington Heights, IL, USA)) for 45 min, followed by pH-sensitive liposomes composed of cholesteryl hemi- rinsing and application of streptavidin-Texas Red (1:250, succinate (CHEMS)/dioleoylphosphatidylethanolamine Gibco-BRL) for 15 min. Exposure times for both film and (DOPE)/iron transferrin-N-glutaryl phosphatidyl- prints were held constant for all sections. ethanolamine (NGPE) were prepared by detergent dialy- sis method. Briefly, lipid films composed of DOPE and Transferrin–enzyme conjugates CHEMS (6:4; mol/mol) were formed under a nitrogen Biotin–transferrin was prepared by reacting iron trans- stream and suspended in 20 mm Hepes/150 mm NaCl, ferrin with amino-reactive biotin containing a long- pH 8.0. The lipid suspension was sonicated with a bath chain spacer arm (succinimidyl-6-(biotinamido) hexano- sonicator and the pH was adjusted to 8.0. Conjugation of ate (Pierce)) following the manufacturer’s instructions. transferrin to NGPE was performed as previously 42 Free biotin was removed by three rounds of concen- described. Briefly, NGPE dissolved in CHCl3 was dried tration and resuspension in a microconcentrator with N2 gas and vacuum desiccated. The dried NGPE (molecular weight cut-off 30 kDa, Amicon), and the was then solubilized in 0.15 m octyl-glucoside (OG) at a degree of biotin incorporation was determined by NGPE:OG ratio of 0.06:1 (mol/mol) in 2-(N-morpholino) HABA-dye (2-(4′-hydroxyazobenzene) benzoic acid; ethansulfonic acid (MES) buffer (10 mm MES/150 mm Pierce) assay following the manufacturer’s instructions. NaCl, pH 5.0). 1-Ethyl-3(3-dimethylaminopropyl) car- Biotin–alpha-bungarotoxin was obtained commercially bodiimide (EDC) and N-hydroxysulfosucciniimide (Molecular Probes, Eugene, OR, USA). To assess the (Sulfo-NHS) were added to the above mixture to a ability of modified ligands to bind with high affinity to NPE/EDC/Sulfo-NHS ratio of 1:50:20 (mol/mol) and their myotube receptors, binding competition assays then incubated for 10 min at room temperature. The mix- were performed with both biotin-derivatized ligands on ture was neutralized with 100 mm HEPES buffer and 1 dystrophin-deficient myotubes. Radiolabeled native N NaOH to pH 7.5. Transferrin containing a trace amount Ligands for gene delivery to myogenic cells WG Feero et al 673 of 125I-labeled transferrin was then added at a trans- 3 Perales JC, Ferkol T, Molas M, Hanson RW. An evaluation of ferrin:NGPE ratio of 1:15 (mol/mol) and incubated for 8 receptor-mediated gene transfer using synthetic DNA–ligand hat4°C with gentle stirring. NGPE derivatized trans- complexes. Eur J Biochem 1994; 226: 255–266. ferrin (1/25th of total lipid by weight) and octyl gluco- 4 Wang CY, Huang L. pH-sensitive immunoliposomes mediate side (final concentration of 200 mm) were then added into target-cell-specific delivery and controlled expression of a foreign gene in mouse. Proc Natl Acad Sci USA 1987; 84: 7851– the liposomes and the mixture was dialyzed at 4°C m 7855. against 20 m Hepes/150 mM NaCl, pH 8.0. After dialy- 5 Wu GY, Wu CH. Receptor-mediated in vitro gene transform- sis for 48 h, the liposomes were extruded through a 0.1 262 ␮ ation by a soluble DNA carrier system. J Biol Chem 1987; : m polycarbonate filter to obtain liposomes of uniform 4429–4432. size distribution. Subsequently, the liposomes were sep- 6 Wilson JM et al. Hepatocyte-directed gene transfer in vivo leads arated from free transferrin by column chromatography to transient improvement of hypercholesterolemia in low den- on BioGel A1.5 M (Bio-Rad). 3H-labeled cholesteryl hex- sity lipoprotein receptor-deficient rabbits. J Biol Chem 1992; 267: adecyl ester (DuPont, Boston, MA, USA) was used to 963–967. monitor the lipid, while 125I transferrin was used to fol- 7 Wilson JM et al. A novel mechanism for achieving transgene low the transferrin. 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