Biomaterials for Gene Delivery: Atelocollagen-mediated Controlled Release of Molecular Medicines

Takahiro Ochiya¹*, Shunji Nagahara², Akihiko Sano², Hiroshi Itoh³, and Masaaki Terada¹

¹National Cancer Center Research Institute, 1-1, Tsukiji, 5-chome, Chuo-ku, Tokyo 104-0045, Japan

²Formulation Research Laboratories, Sumitomo Pharmaceuticals Co., Ltd., 3-45 Kurakakiuchi 1-chome, Ibaraki-shi, Osaka 567-0878, Japan

³Koken Bioscience Institute, 2-6, Okubo 2-chome, Shinjuku-ku, Tokyo 169-0072, Japan

*Address correspondence to this author at the Section for Studies on Metastasis, National Cancer Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan; Tel: +81-3-3542-2511 x 4420; Fax: +81-3-3541-2685; Email: tochiya@ncc.go.jp

Abstract: Over the last decade, increasing attention has been paid to the development of systems to deliver drugs for long periods at controlled rates. Some of these systems can deliver drugs continuously for over one year. However, little effort has been given to developing systems for the controlled release of nucleic acids. Recently, a novel gene transfer method which allows prolonged release and expression of plasmid DNA in vivo in normal adult animals was established. In this system, a biocompatible natural polymer such as collagen or its derivatives acts as the carrier for the delivery of DNA vectors. The biomaterial carrying the plasmid DNA was administered into animals and, once introduced, gradually released plasmid DNA in vivo. A single injection of plasmid DNA/biomaterial produced physiologically significant levels of gene-encoding proteins in the local/systemic circulation of animals and resulted in prolonged biological effects. These results suggest that the biomaterials carrying plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Furthermore, the biomaterials can be removed surgically, minimizing the effect of gene products if some unexpected side effects should be observed after application. The application of these systems to expand the bioavailability of molecular medicine, including antisense oligonucleotides and adenovirus vectors, and to aid in stem cell transplantation in the context of DNA-based tissue engineering will be discussed.

Present State and Problems of Gene Delivery Technology

     Revolutionary information to elucidate all biological phenomena at the molecular level has been brought about by the recent rapid development of molecular biology and the progress of the human genome project. Tissue engineering [Langer, 1993; Mooney et al., 1999], drug delivery systems (DDS) [van Ommen et al., 1999; Heinemann, 1999; Hoffman, 1995; Langer, 1990], and gene therapy [Anderson, 1998; Miller, 1992] have rapidly grown with the aid of such scientific background and are now spotlighted as the most hopeful research fields of medicine and biotechnology.

     Among these new research fields, gene therapy is considered to be a 21st-century technology based on advancements in molecular biology provided by the human genome project. From the viewpoint of pharmacotherapy, the approach of gene therapy has focused on the delivery of macromolecular drugs such as nucleic acids to target organs and tissues. What we now need, however, is the development and application of DDS indispensable for the establishment of effective means of gene therapy using gene-related medical supplies. In spite of such a common understanding, emphasis has been placed only on the development of the vector to obtain the final efficiency of gene expression, but its application from the pharmacokinetical aspect has been disregarded. Molecular medicines are new materials packed with genetic information, and their fundamental pharmacokinetics, after their systemic or local administration, are controlled by physico-chemical properties such as molecular weight and charge. Therefore, it is desirable, for the development of DDS technology, to design gene drugs with sufficient consideration of the factors influencing their pharmacokinetics. Several synthetic materials, including cationic liposomes such as DOTMA [Ren et al., 2000], DC-chol [Nomura et al., 1999], and DOSPA [Hofland et al., 1997], cationic polymers such as dendrimers [Trunen et al., 1999], PLL [Wolfert et al., 1996], and PEI [Boussif et al., 1995], and HVJ-liposome [Kaneda et al., 1999] have been generated and successfully used for transferring DNA into mammalian cells [Han et al., 2000] both in vitro and in vivo. However, these synthetic materials are often less efficient and are highly toxic after repeated use; as a result, prolonged in vivo usage is not allowed. In this review article, we are focusing on natural polymers as DNA carriers because they are considered to be biocompatible for human clinical use.

What Does “Gene Delivery Technology by Biomaterials” Mean?

     The aim of gene therapy is to treat diseases involving deficient or mutated proteins by delivering genes that encode intact proteins to target cells and making them express there. This is thus expected to be a new way to treat refractory diseases such as congenital diseases, cancer, and AIDS. Within this strategy, plasmid vectors and adenovirus vectors are widely used to express the target gene transiently without expecting its insertion into the chromosome for the treatment of those diseases. However, the vector, when given by the usual method, is inactivated and degraded immediately after its contact with cells. Therefore, the vector method is not suitable for the treatment of a disease requiring gene expression over several weeks or more, during which the copy number of the administered gene decreases through dilution by cell division and intracellular degradation occurs. To overcome this shortcoming, repeated administration of the virus vector is required. This, however, imposes a heavy burden on the patients because the delivery of amounts of genetic material in excess of its physiological concentration causes serious side effects [McElvaney, 1996; Kaplan et al., 1997]. In order for gene therapy to be applicable in clinical medicine, it is imperative that a suitable method for stable controlled release of the required amount of the vector delivered over the desired period of time be developed. Moreover, it would be desirable to be able to stop delivery and expression of the gene as soon as it is decided to stop the treatment and to minimize the significant side effects of this type of therapy. An approach to such a system is the utilization of materials with high molecular weight, which are derived from the human body as a carrier of gene-transferring vectors. Since biomaterials exhibit favorable biological properties [Hoffman, 1992], that fact has caused great attention in the biomedical fields. In this sense, the following properties are suitable for combining molecular medicine with biomaterials (Fig. 1): (I) The materials are biocompatible and have the least antigenicity and toxicity. (II) The materials are soluble and easily mixable and fusible with various gene vectors. (III) The materials are biodegradable and gradually releasable over an appropriate concentration range and within a time schedule. (IV) The materials can be sealed from the attack of enzymes in vivo and retain their structures and activities, as rapid degradation of DNA is a serious problem in gene therapy. (V) The materials have plasticity; in other words, the liquid phase easily converts to a solid one when they are delivered to patients. (VI) The materials can be easily removed from the patients’ bodies with minimum invasion.

Polymer-based Gene Delivery Systems

     (Table 1) shows a list of the biomaterials which satisfy the conditions described above from the point of view of safe implantation of the gene vector in vivo and controllability of the prolonged release of gene material at a fixed concentration and on a definite time schedule. In regard to the gene delivery of polymer-based biomaterials into the body, an attempt using EVAc (polyethlene vinyl co-acetate) was made [Jong et al., 1996]. Jong et al. showed that DNA was released from the EVAc without degradation and retained the ability to transfect cells in vitro. Saltzman’s group also showed that both small and large DNA molecules were encapsulated and successfully released from EVAc matrices [Luo et al., 1999]. However, the studies ended up only in a model experiment in vitro, and the dynamic phase in vivo remained unexplored. The self-assembly of polymer “microspheres” that can carry a variety of medical materials including plasmid DNA has also been described [Mathiowitz et al., 1997]. Orally administered biodegradable poly(FA:SA) 20:80, which are polyanhydride copolymers of fumaric and sebacic acid with highly adhesive properties, allowed gene delivery to transfer biologically active molecules to the body. Alginate, a naturally occurring biopolymer extracted from brown algae (kelp), has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of biological agents including nucleic acid [Alexakis et al., 1995; Aggarwal et al., 1999]. In fact, biodegradable alginate microspheres were used as a delivery vesicle for DNA based vaccines [Mittal et al., 2001]. They showed that mucosal immunization route lead to a significant immune response against LacZ-coding DNA encapsulated into alginate.

Fig. (1). Biomaterials suitable for DNA delivery.

     The efficient delivery of growth-promoting genes locally in a sustained manner is important for effective tissue regeneration. Dr. Mooney’s group reported that the in vivo delivery of a plasmid DNA encoding a platelet-derived growth factor gene using a polymer matrix, poly(lactide-co-glycolide), enhanced matrix deposition and blood vessel formation in the developing tissue [Shea et al., 1999; Murphy et al., 1999]. Plasmid DNA carrying a fragment of the human parathyroid hormone gene was carried into target tissue for its regeneration by a polymer matrix sponge called a gene-activated matrix (GAM) [Bonadio et al., 1999; Bonadio et al., 2000]. Implantation of GAM at bone injury sites was associated with the retention and expression of plasmid DNA for a longer period and resulted in reproducible new bone tissue regeneration. Another challenge comes from the delivery of cytokine genes for the treatment of disease by the use of poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA), which is a biodegradable analogue of poly(L-lysine)[Lim et al., 2000; Maheshwari et al., 2000]. This biomaterial did not show cytotoxicity, possibly because of its degradability, and its biocom-patibility allowed significant enhancement of cytokine gene expression at mRNA and protein levels both in vitro and in vivo. Imidazole-containing polymers also seem an interesting and excellent method [Pack et al., 2000]. This polymer allows efficient release of the DNA/polymer complex from endocytotic vesicles into the cytoplasm, a notion that is of primary importance for successful drug delivery and tissue engineering [Langer, 1999].

    DNA delivery technologies by a chitosan, a natural cationic polysaccharide derived from naturally occuring chitin in crab and shrimp shells by deacetylation has previously used as a pharmaceutical excipient in oral drug formulation as well as suture and wound dressing materials [Felt et al., 1998], and its derivatives have been reported. Recent reports suggest its usefulness for a colon-specific drug delivery system [Tozaki et al., 1997; Genta et al., 1999]. The observations were that the highly purified chitosan fractions were neither toxic nor haemolytic and that they had the ability to increase the complexity of DNA and protect against nuclease degradation [Quong et al., 1998; Quong et al., 1999]. Furthermore, low-molecular-weight chitosan can be administered intravenously without accumulation in the liver [Richardson et al., 1999], indicating its potential for therapeutic use. In fact, an oral allergen-gene immunization with chitosan-DNA nanoparticles is effective in modulating murine anaphylactic responses and indicates its prophylactic utility in treating food allergies [Roy et al., 1999]. The presence of a pH-sensitive endosomolytic peptide enhanced the levels of reporter gene expression when the chitosan/DNA complex was administered in the intestinal tissues, although levels of expression remained low [MacLaughlin et al., 1998]. In defined conditions, plasmid DNA conjugated with chitosan produced small discrete particles with a diameter of approximately 50-100 nm and higher transfection efficacy to specific cells than did PEI [Erbacher et al., 1998]. The self-aggregates of deoxycholic acid-modified chitosan allowed efficient DNA transfer to COS-1 cells [Lee et al., 1998]. These results suggest that chitosan is a strong candidate non-viral vector for effective gene delivery.

Fig. (2). Schematic Representation of Atelocollagen Molecule.

     A DNA delivery system using gelatin was developed by several research groups and has been shown to have practical applications. Controlled gene delivery by DNA-gelatin nanospheres has been reported [Truong-Le et al., 1998; Leong et al., 1998; Truong-Le et al., 1999]. These authors used cross-linked gelatin to control the release of the nucleic acid and keep its matrix structure. This material has several potential advantages for gene delivery in that the gelatin nanosphere prevents DNA degradation in serum and allows cell targeting and co-delivery of other bioactive substances. However, chemical modification of natural polymers to improve its ability for gene transfer results in some problems regarding safety. Therefore, the use of a natural polymer without any modifications such as cross-linkage is highly desired for practical applications.

Gene Delivery Technology by Atelocollagen

     As the most potent candidate which satisfies the condition shown in (Fig. 1), we chose atelocollagen, a decomposition product of type I collagen derived from the dermis of cattle. Collagen is a fibrous protein of connective tissue (such as the dermis of the skin) that plays an important role in the maintenance of the morphology of tissues and organs [Ramirez et al., 1988]. Collagen (toropocollagen) consists of 3 polypeptide chains and is helical in structure. It is a rod-like molecule with a length and diameter of 300 nm and 1.5 nm, respectively (Fig. 2). The molecule has an amino acid sequence called "telopeptide" on both its N- and C-terminals, which include most of the antigenicity of collagen. Atelocollagen is obtained by pepsin digestion [DeLustro et al., 1986] and is free from telopeptides, indicating that it has low immunogenicity [Stenzel et al., 1974]. (Table 2) shows its characteristics as a medical material. Collagen, as well as atelocollagen, is widely used in biodegradable medical supplies such as suture materials, hemostatic drugs, wound coatings, and intradermal injection medicine for the repair of collapsed areas of the skin [Miller et al., 1964; Cameron et al., 1978; Doillon et al., 1986; Kamer et al., 1984]. Plasticity, which is one of the features of atelocollagen, is very important for gene delivery in vivo [Fujioka et al., 1995]. This plasticity is based on its solubility: atelocollagen is soluble at a lower temperature but solidifies to refibrillation at a temperature over 30^oC (Fig. 3). Therefore, atelocollagen is used not only as a fluid or gel, either of which can be injected locally, but also as solid matter such as beads, sponges, membranes, and cylinders. This means that atelocollagen can be processed to profile all spaces in the body as well as organs and blood vessels when it is used as an internal implant. Thus, a gene delivery method using atelocollagen would solve issues of site specificity and target gene transfer. Moreover, atelocollagen implanted in the body is gradually biodegraded. Therefore, a gene included in a vector can be expected to be released gradually and with appropriate control of the concentration, shape, and dose of the gene as well as of the site of injection.

Preparation of the Minipellet

     Current gene therapy shows a strong trend to deliver a “naked” or “modified” plasmid DNA preparation to the body instead of a DNA virus vector to obtain a substantial therapeutic effect [Wolff et al., 1990; Davis et al., 1993; Manthrope et al., 1993; Tripathy et al., 1996; Levy et al., 1996], owing to the development of gene delivery reagents such as liposomes.

Fig. (3). Solution–to-Gel Transition of Atelocollagen.

This method is mainly based on the administration of plasmid DNA into the muscle, where the DNA remains as an episomal form for a certain period and thus the encoding protein is produced in that organ as a factory. Actually, however, “naked” plasmid DNA is more rapidly degraded in vivo than was once expected, and its expression is transient (Fig. 4). This means that the effect of plasmid DNA is restricted, as is that of the DNA virus vector.

Fig. (4). Pharmacokinetic feature of biomaterial-based DNA delivery system. Naked plasmid DNA, adenovirus vector and protein were short-lived in vivo. Biomaterial-based DNA delivery showed a sustained biological effect. Furthermore, biomaterial allowed stop gene delivery at desired time (*1). If desired, repeat administration (*2) of gene product can be achieved.

     We have developed a new technology in which plasmid DNA is embedded in atelocollagen [Ochiya et al., 1999], by which the quantity and period of the gene expression are well controlled in vivo (Fig. 4). For the purpose of a model experiment using animals, we prepared atelocollagen of a cylindrical form with a diameter of 0.6 mm, enclosed 50-100µg of plasmid DNA inside the former, and administered it to the femoral muscle as a target site (Fig. 5), left panel).

     This preparation is called “Minipellet” and can be implanted into the body using a convenient instrument if an appropriate size is chosen. Plasmid DNA embedded in the Minipellet remained intact for a certain period in vivo and appeared to be released gradually both locally and systemically (Fig. 5), right panel), indicating that atelocollagen may be useful in avoiding rapid degradation of plasmid vectors in vivo, thereby enabling prolonged treatment. We used a plasmid vector in our experiment in which a cDNA fragment of the HST-1/FGF-4 gene of the human fibroblast growth factor (FGF) was included as an easily detectable biological marker in vivo instead of reporter genes such as LacZ and GFP. Since this gene delivery results in an increase in platelet counts in the periphery blood [Konishi et al., 1995; Konishi et al., 1996; Takahama et al., 1999], the effects of gene delivery and the validity period in an animal body can be easily evaluated by simple sampling of the blood.

Optimum Composition Suitable for Controlled Release of Plasmid DNA

     In general, plasmid DNA can be formed as a Minipellet through a freeze-drying process. The process is widely used in the manufacture of medical supplies, since it activates heat-unstable substances such as proteins rather than biologically active ones. Unexpectedly, however, we found that plasmid DNA decomposed during this freeze-drying process. At present, the clinical approach to gene therapy remains immature, with attention being paid only to how much plasmid DNA is expressed but not to how efficiently it is expressed or what configuration it takes. Plasmid DNA takes the configuration of a supercoil unless the DNA chain is not decomposed, while it is changed to an open circle or a linear chain when it is severed. It is also known that the structural change of DNA plasmid results in a marked decrease in its expression efficiency. Little information is available for the rescue of such plasmid degradation by the freeze-drying process except for the encapsulation in polymer matrixes such as poly((D), (L)-lactic-co-glycolic acid) (PLGA) [Ando et al., 1999]. To prepare medical supplies from plasmid DNA, it is necessary to develop technology to prepare them in such a way that the tertiary structure of the DNA is stably maintained. On the other hand, plasmid DNA from the therapeutic preparations would be pharmaco-logically regulated at a velocity suitable for its delivery to and expression in the target cells. In our case, the Minipellet is a preparation in which the plasmid DNA is embedded in a collagen matrix of highly dense structure by adding glucose. Glucose not only suppressed the decomposition of plasmid DNA in the freeze-drying process but also promoted the release of plasmid DNA from the Minipellet in vivo. It was also found that amino acids such as glutamine had a similar effect as a protector of plasmid DNA during freeze-drying and other processes (unpublished observations). Moreover, we also found that plasmid DNA molded as the Minipellet was stable for at least 3 months at room temperature and for over half a year at 4^oC.

Minipellet Implant Into Experimental Animals and Its Effect

Fig. (6). Kinetic demonstration of gene delivery with the Minipellet. Serum protein concentration (A) and platelet count (B) showing that biomaterial-based plasmid DNA delivery allowed long-term release and expression of DNA in vivo.

     (Fig. 6) shows the outline of the results with experimental animals. We injected the Minipellet into the muscle along its fiber in the femoral region of an ICR mouse and then examined how much of the plasmid DNA was released into the body and how well the gene expression was regulated after peripheral blood and various organs were sampled at fixed intervals by the PCR method (Fig. 6A). In a control experiment using plasmid DNA alone, the release of DNA to the peripheral blood lasted 7 days after its administration. In the experiment with the Minipellet, it persisted over a period of 40 days, notwithstanding the fact that the first appearance of DNA in the blood with the Minipellet was at the same time as that with plasmid DNA alone (6 hours later). Moreover, plasmid DNA was detected in the muscle of the injection area 60 days later in the case of the Minipellet. These facts suggest that intramuscular administration of the Minipellet might result in longer release of DNA than that of plasmid DNA alone, which had so far been used, and that the Minipellet protected DNA from biodegradation. It is anticipated that DNA released from the Minipellet in vivo is combined with atelocollagen. Upon examining how fast plasmid DNA is degraded in the serum in vitro, it was observed that atelocollagen actually inhibited digestion of the plasmid DNA by nuclease (Fig. 7). We observed the increase in platelet counts for more than 70 days when the Minipellet was injected, reflecting the longer existence of plasmid DNA with the Minipellet than with plasmid DNA alone (Fig. 6B). These results suggest that atelocollagen plays an important role in gene therapy as a biocom-patible material, increasing the effectiveness of plasmid DNA in vivo.

Consideration of Safety in the Use of a Vector for Gene Therapy

 It is desirable to have the ability stop the delivery and expression of the DNA included in the vector as soon as possible when it is considered that the treatment must be terminated, depending on the type and symptoms of the genetic diseases. It is also desirable to reduce the side effects of the therapy on the patient. Our Minipellet can be easily recognized in vivo from its cloudiness and solidity and surgically removed within 2 weeks of its delivery (Fig. 5), right panel). Accordingly, the Minipellet system can be used with a high degree of safety in cases in which side effects appear at an early period and the treatment must be discontinued immediately. However, it is difficult to remove the pellet completely from the muscle using a pair of tweezers because atelocollagen is biodegradable and swelling and fragmentation follow for a period of over two weeks after the injection. Therefore, it is necessary to develop a device that allows safe use of DNA vectors and easy removal from the implanted position with minimum damage when the patient himself desires to have it removed.

Fig. (7). Atelocollagen blocks plasmid DNA from degradation by nuclease attack. Naked plasmid DNA (supercoiled, indicated by arrow) was digested with nuclease in 15 minutes. Atelocollagen/DNA showed resistance against nuclease attack even in 60 minutes in a 10% serum solution.

Prolonged Release of Adenovirus Vector In Vivo

     Adenovirus vectors are currently the most widely used gene transfer method in gene therapy, partly because they form an efficient system for the delivery and expression of foreign genes into several types of mammalian cells in vitro [Kozarsky et al., 1993], allowing for the systemic delivery of foreign DNA [Ragot et al., 1993; Le Gal La Salle et al., 1993; Brody et al., 1994] as well. However, the adenovirus vectors remain in an extrachromosomal location and are replication-defective, resulting in transient expression; this has caused the phase I clinical trial to have only limited success. Thus, strategies to increase efficiency of virus vectors are desirable. The potential use of polymer materials to increase virus vectors was assessed. Siemens et al. evaluated the effect of several polymer matrices to deliver the canarypox virus ALVAC to the cells of the murine prostate cancer cell line and found that a gelatin sponge matrix proved to be the most effective vehicle for delivering viral vectors in vitro and in vivo [Siemens et al., 2000]. To accomplish low-dose administration of virus vectors, a method to formulate recombinant adenovirus vectors in biodegradable microspheres was developed [Davidson et al., 1997]. These authors showed that poly (lactic-glycolic) acid (PLGA) microspheres containing recombinant adenovirus allowed viable viruses to be released for periods longer than 10 days. The encapsulated adenovirus also showed diminished immunogenicity in vivo [Beer et al., 1998; Matthews et al., 1999]. In this report, mice immunized with encapsulated recombinant adenovirus vectors showed a greater than 45-fold reduction in anti-adenovirus titers relative to non-encapsulated vectors.

Fig. (8). Administration of atelocollagen implant carrying virus vector into animals. Equal volumes of adenovirus solution and 0.4% of the atelocollagen implant solution were mixed with gentle pipetting at a cold temperature. The mixed solution was intraperitoneally, subcutaneously or intramuscularly injected into mice. Once introduced into animals, it becomes solid, releases adenovirus gradually and produces reporter proteins longer than does adenovirus transfer alone

     (Fig. 8) shows the outline of the delivery of an adenovirus vector with atelocollagen into the body. In this case, the adenovirus was suspended in a buffer solution and mixed with liquid atelocollagen at 4^oC. Here, we show the result of a model experiment using a recombinant adenovirus carrying AdexHST-1 [Sakamoto, et al., 1994], the gene for the human fibroblast growth factor, which promotes the growth of platelets. Our experimental design was based on the view that this system would ease the monitoring of biological activity after its delivery to the body of an animal. To substantiate this, we first determined the concentration of atelocollagen and the amount of virus needed for fulfilling the desired effect of the therapy and biological activity individually. The controlled release of adenovirus was dependent on the atelocollagen concentration in such a way that the virus was not sufficiently released unless the atelocollagen concentration was less than 1 %. The optimum for doubling platelet counts in the peripheral blood was obtained when AdexHST-1 (10⁹ pfu) was mixed with atelocollagen so that the latter's final concentration was adjusted to be 0.2%. The liquid mixture of atelocollagen and adenovirus was solidified at body temperature, stayed around the injection site or liver, and was biodegraded; in addition, it was gradually released over a long period when it was injected into the peritoneal cavity of the animal. Adenovirus vectors embedded in atelocollagen were kept viable for a certain period in vivo and seemed to be released gradually, indicating that the atelocollagen implant may be useful in avoiding rapid degradation of adenovirus vectors in vivo and therefore allow prolonged treatment. This is partly because the atelocollagen may protect from the attack of protease activity or nuclease activity against adenoviruses embedded in the implant in vivo. Indeed, our study revealed that a single injection of the atelocollagen implant carrying AdexHST-1 allowed continuous transgene expression for at least 65 days without additional administration of adenoviruses (Fig. 9), suggesting that our system may reduce the side effects of large dosages and repeated treatment of virus vectors.

“In-out” Strategy Using AtelocoL-lagen Implants

     Recently, several attempts were made to regulate transgene expression in vivo, including temperature-sensitive SV40 large T antigen [Jat et al., 1991], the bacterial lac repressor/operator system [Deuschle et al., 1990], Cre recombinase-lox strategy [Lasko et al., 1992], and the bacterial tetracycline-resistance operon regulatory system [Efrat et al., 1995]. Another interesting approach to regulate gene expression is the use of adenovirus expressing the site-specific Cre recombinase system [Kanegae et al., 1995]. These systems have the possibility of turning transgene expression on-off during the course of treatment of diseases by means of human gene therapy. However, substantial efforts are required for the strict control of transgene expression in vivo. Here, we presented a simpler and safer method to control the transgene expression of adenovirus vectors in vivo using an atelocollagen implant as a carrier. Once introduced into animals, the atelocollagen/adenovirus conjugate becomes solid, remaining in the body for a certain period. As a means to control gene expression of injected adenovirus vectors, the atelocollagen implant carrying AdexHST-1 was removed 3 days after the in vivo administration. As shown in (Fig. 9A), serum HST-1 levels were reached in a maximum period of 12 days after the administration and then rapidly returned to normal at day 20. The platelet count increased for 10 days after the removal of the implant and then dropped rapidly and returned to normal at 34 days after the administration (Fig. 9B). This result indicates that the surgical removal of the atelocollagen implant carrying adenovirus vectors may enable us to regulate the duration of the gene expression as well as the biological effects of the administered genes in vivo. It is suggested that the present method can be used to effectively regulate the serum concentration of other administered growth factors or cytokines.

Fig. (9). “In-out” strategy uses atelocollagen implant. (A) Production of HST-1 protein by atelocollagen implant: serum HST-1 levels were kinetically analyzed in mice (n=10) with administered AdexHST-1 with (m) or without (l) 0.2% atelocollagen implant. In some experiments, administered implants were surgically removed from the peritoneal cavity 3 days after the implantation (o). (B) Kinetic analysis of the platelet count: mice with atelocollagen implant carrying AdexHST-1 were analyzed for their platelet count (m). As a control, AdexHST-1 alone was injected (l). In a separate experiment, the implanted atelocollagen carrying AdexHST-1 was retrieved from the peritoneal cavity 3 days after the injection and then the platelet count was analyzed kinetically (o, n=6)

Atelocollagen Allows Repeated Administration of Adenovirus Vectors

     We obtained further important information during the examination of this method: an adenovirus vector embedded in atelocollagen delivered into the body escaped an attack by neutralizing antibodies and thus enabled continuous administration of the adenovirus to administer continuously (Fig. 10), which is usually impracticable. When an adenovirus vector is applied clinically, significant obstacles such as cell-mediated immunity and humoral immunity responses of the host to adenovirus itself [Yang et al., 1995-a] should be removed, as they result in difficulty in repeating the administration of the virus vector, which is needed for any long-term treatment. For this purpose, the following strategies have so far been developed: inhibition of the immunological responses of the host by interferon or interleukin [Yang et al., 1995-b; Smith et al., 1996]; acquisition of “oral tolerance” by continued administration to the oral cavity [Ilan et al., 1998; Kagami et al., 1998] and the use of adenovirus vectors of more than 40 different serotypes [Mack et al., 1997; Mastrangeli et al., 1996]. Our newly developed method for gene delivery to the body by atelocollagen can be used for repeated administration to animals who have neutralizing antibodies to the adenovirus by simply conjugating with atelocollagen before the second or third administration of adenoviruses. One possible explanation is that the released adenovirus is still associated with atelocollagen and thereby protected from the attack of the neutralizing antibody, resulting in sustained circulation of the adenovirus in the peripheral blood (Fig. 11). In fact, in vitro experiments showed that the adenovirus conjugated with atelocollagen was protected for a longer period against destruction by serum with the neutralizing antibody. Thus, it can be concluded that application of our system to increase the bioavailability of adenovirus vectors is interesting to broaden the efficacy of gene therapy.

Fig. (10). Repeat administration of adenovirus vector. Animals were first given adenovirus vector alone. Thirty-eight days after the first administration, mice were given second adenovirus administration with either adenovirus alone (n) or adenovirus conjugated with atelocollagen(l). Serum neutralizing antibody against adenovirus in mice was detected 7 days after the first adenovirus injection

Applications of Atelocollagen to Antisense Therapy

     The goal of our new gene delivery technology is to use it for a gene expression vector with biomaterial as a carrier to the whole body and/or the target organs for gene therapy at its optimum concentration and time point and obtain the best therapeutic effect. Such a basic concept has already been accepted as a matter of common knowledge in the protein transport preparations in the field of DDS. We believe that our study is one of the first to link conventional DDS with recently developed gene therapy. It is also confirmed that our system is actually applicable for the delivery of DNAs related to antisense technology. Anti-sense drugs are especially hopeful antivirus and anticancer drugs,and their clinical applications have now started [Akhtar et al., 2000; Ma et al., 2000; Dropulic et al., 1994].

  A critical factor for the therapeutic application of antisense strategy is an effective methodology for the delivery of oligodeoxynucleotides (ODNs) in vivo. Previous studies revealed that phospholothioate ODNs are distributed broadly in tissues after intravenous and intraperitoneal administration and exclusively secreted out into urine, which suggests that the ODNs have a short in vivo life [Agrawal et al., 1995]. It has been reported that an avidin-biotin system [Bonado et al., 1992], liposomes [Kanamaru et al., 1998; Ochiya et al., 2000], and cationic amphiphiles [DeLong et al., 1999] protect antisense ODNs, and this property may be useful for delivery of antisense drugs to tissues in vivo or to cultured cells in vitro. However, these compounds was also associated with cytotoxicity, suggesting that optimization of these formulations will be necessary to achieve a safer and more efficient delivery of the ODNs in vivo. Here we have addressed a simple and safe method to control the delivery of ODNs in vivo using an atelocollagen implant as the carrier material. ODNs embedded in the atelocollagen were kept intact for a certain period in vivo and seemed to be released gradually, indicating that an atelocollagen implant may be useful in avoiding rapid degradation of ODNs in vivo and therefore allow controlled release as well as prolonged treatment. This is partly because the atelocollagen may protect from the attack of nuclease activity against ODNs embedded in the implant in vivo. At the same time, because the atelocollagen implant remained a solid mass that could be handled easily, the implant becomes an attractive carrier for the removal of antisense ODNs when it is desirable to stop the treatment of gene therapy. Furthermore, the major advantage of the atelocollagen is that it is less toxic and also biodegradable, resulting in low immunogenicity to the host. Another interesting approach through atelocollagen implantation in vivo is the targeting administration of ODNs. In this regard, the solid state of atelocollagen in vivo also has great potential for the tissue-specific transportation of ODNs. These results suggest that atelocollagen implants may enhance the clinical potency of antisense ODNs, facilitating a more effective and safer use of antisense strategy for gene therapy.

Application of Atelocollagen to Stem Cell Transplantation in Regeneration Medicine

     Recent progress in tissue engineering has enabled us to produce functional artificial tissues from embedded cells into polymeric scaffolds [Langer, 1993]. However, these scaffolds cannot include the bioactive molecules, including DNAs, which are required to feed the embedding cells. Therefore, the development of a biomaterial-based gene transfer method that combines gene therapy and tissue engineering to promote tissue regeneration is anticipated. In this connection, atelocollagen possibly contributes to stem cell transplantation as a carrier of the cells. As mentioned above, collagen primarily works as a scaffold of cell adhesion. The collagen molecule promotes the seizing of various stem cells, each of which is differentiated to specific organs and tissues such as the nerve, pancreas, muscle, capillary endothelium, and blood cells. Currently, these phenomena have become relevant, and the combination of biomaterial-based gene delivery with stem cell technology can present a suitable transplantation system. Collagen molecules can be reconstituted with the same three-dimensional structure as biological tissues inside its structural fiber mesh. Thus, they may contribute to produce artificial tissues in such a way that intercellular adhesion or extracellular matrix-based cell-cell interaction is completely reproduced. Such ability of atelocollagen to induce the controlled release of the gene from the expression vector is expected to enable stem cells not only to produce the molecules needed for their maintenance and differentiation and tissue regeneration ability, but also supply them stably over a long period. This DNA-based tissue engineering approach also provides artificial tissue for transplantation with higher levels of accomplishment and self-support [Murphy & Moony, 1999]. Therefore, it can be foreseen that the atelocollagen technology will contribute greatly to regeneration medical treatment as well as to gene therapy (Fig. 12).

     Another interesting challenge was met by the use of a gelatin-based resorbable sponge [Ponticiello et al., 2000]. By culturing adult mesenchymal stem cells embedded in a gelatin sponge for 21 days in vitro in a defined medium supplemented with TGF-beta 3, these authors produced a cartilage-like extracellular matrix. When implanted in an osteochondral defect in the rabbit femoral condyle, gelfoam cylinders were observed to be very biocompatible, with no evidence of immunological rejections. Using the gelform resorbable gelatin sponge as a DNA delivery vesicle indicates a promising candidate as a carrier matrix for stem cell-based regeneration therapies.

     To achieve excellent polymer-based tissue regeneration in vivo, controlled growth factor delivery is needed. To date, there have been many attempts to control drug release in vivo by using external stimuli such as pH [Chen et al., 1995], an oscillating magnetic field [Edelman et al., 1987], and ultrasound [Mitragorti et al., 1995]. Recently, a mechanical stimuli-mediated drug delivery system has been developed [Lee et al., 2000]. This novel system successfully presented that vascular endothelial growth factor (VEGF)-loaded hydrogels subjected to physiological mechanical signalling allowed upregulation of VEGF to promote blood vessel formation. All these approaches could allow precise control of drug delivery in vivo, and might be applicable to regulate the release dose and rate of molecular medicines.

 

Economic Efficiency and Market-ability of Gene Delivery Techno-logy by Biomaterials

     The clinical application of gene therapy has yielded various excellent virus vectors and tested their efficiency and safety. However, strict quality controlled vectors, including thermal treatment, are required before they can be used on patients, since the vector is susceptible to inactivation and special facilities are needed for their manufacture. Moreover, a strictly close cooperation system should be established between the clinicians who use the products and the researchers who offer them. Therefore, many difficulties remain to be overcome for the generalization of the clinical uses of gene products that remain safe and effective. It is also quite natural to require ample funds to clear these conditions. In the meantime, cationic or lipid liposome is being examined as a nonviral vector, but plasmid DNA products using these vectors should be usually prepared immediately before delivery owing to their instability. The delivery and expression efficiencies of DNA in cells by such plasmid DNA products are largely dependent on the doctors’ skill and conditions of the facilities. These have generally hampered the wide use of the products for gene therapy. The DNA delivery system by atelocollagen, especially that with the Minipellet described in this paper, is considered to enable distribution through the same channel as other medical supplies, since it is stably protected for a definite period at room temperature. The Minipellet can be administered using a convenient instrument, as described before [Fujioka et al. 1995]. In regard to the popularization of our Minipellet system as a strong weapon for gene therapy in the clinical field, this system presents more advantages than the conventional virus and liposome vectors.

     As described above, the practical application of the DDS of protein using the Minipellet has become a step for clinical use. In this Minipellet, bioactive proteins are embedded in a collagen rod with a diameter and length of 1.0 mm and 1.0 cm, respectively [Fujioka, et al. 1998]. It was shown that interferon was gradually released from it into the body for one week and retained its activity when it was implanted into the hypodermis. It was also shown in clinical trials that due to the interferon Minipellet, patients were not only freed from side effects such as feverescence but also from daily visits to the hospital. Based on these facts, it can be concluded that atelocollagen has sufficient qualifications as a gene vector delivery system, like other medical biomaterials.

Summary

     Over the last three decades, DDS applications have become a commercial success. The new targets of DDS are expected to be directed to the focus on gene therapy and regeneration medicine in the 21st century, and they may reach clinical use. It is necessary to improve the delivery system of biomaterials in which the in vivo dynamic state of gene medical supplies can be precisely controlled according to a predetermined pharmacokinetics and a localization to optimize treatment. Accordingly, it is concluded that biomaterial such as atelo-collagen contributes to gene therapy and regeneration medicine as a DNAs delivery system, respectively.

Acknowledgments

     We gratefully thank Ms. Maki Abe and Ms. Masako Hosoda for their excellent technical work. We thank Dr. Eriko Kai for helpful discussions. This work was supported in part by a Grant-in-Aid for the Second-Term Comprehensive 10-Year Strategy for Cancer Control, Health Science Research Grants for the Research on Human Genome and Gene Therapy from the Ministry of Health and Welfare of Japan, and a Grant-in-Aid by Japan Owner’s Association.

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