Molecular Engineering of Matrix-Targeted Retroviral Vectors Incorporating a Surveillance Function Inherent in von Willebrand Factor

Abstract

A major obstacle that limits the potential of human gene therapy is the inefficiency of gene delivery to appropriate sites in vivo. Previous studies demonstrated that the physiological surveillance function performed by von Willebrand factor (vWF) could be incorporated into retroviral vectors by molecular engineering of the MuLV ecotropic envelope (Env) protein. To advance the application of vWF targeting technology beyond laboratory animals, we prepared an extensive series of Env proteins bearing modified vWF-derived matrix-binding sequences and assembled these chimeric proteins into targeted vectors that are capable of transducing human cells. Initially, a dual envelope configuration was utilized, which required coexpression of a wild-type amphotropic Env. Subsequently, streamlined "escort" Env proteins were constructed wherein the inoperative receptor-binding domain of the targeting partner was replaced by the vWF-derived collagen-binding motif. Ultimately, an optimal construct was developed that exhibited properties of both extracellular matrix (ECM)-targeting and near wild-type amphotropic infectivity, and could be arrayed as a single envelope on a retroviral particle. On intraarterial instillation, enhanced focal transduction of neointimal cells (approximately 20%) was demonstrated in a rat model of balloon angioplasty. Moreover, transduction of tumor foci (approximately 1-3%) was detected after portal vein infusion of a matrix-targeted vector in a nude mouse model of liver metastasis. We conclude that the unique properties of these targeted injectable retroviral vectors would be suitable for improving therapeutic gene delivery in numerous clinical applications, including vascular restenosis, laser and other surgical procedures, orthopedic injuries, wound healing, ischemia, arthritis, inflammatory disease, and metastatic cancer.

Critical Stages in the Development of the First Targeted, Injectable Molecular-Genetic Medicine for Cancer

“…Indeed, in the process of metastasis and metastatic tumor formation, both nascent and underlying extracellular matrix (ECM) proteins are characteristically exposed; and it is this characteristic exposure of one particular class of ECM proteins, the ubiquitous and determinative collagens (i.e., collagen patefacio, from Gordon and Hall, 2009), that now forms the basis for disease-seeking (or Pathotropic) tumor targeting. By conceptually grasping the physiological surveillance function that is inherent in the von Willebrand blood coagulation factor (vWF), which normally guides platelets to the sites of vascular injuries, and then physicochemically transposing a synthetic derivative of this physiological surveillance function, via genetic engineering, onto the surface of a nanoparticle-sized gene delivery vector (Hall et al, 2000;Gordon et al, 2000Gordon et al, , 2002, the fields of molecular biotechnology and nanotechnology converged to enable the medical oncologist to reach beyond the mere coverlets of the proverbial bedside and to expose the very fabric of the nature of the metastatic disease process Hall, 2005, 2007). It is the advent of pathotropic targeting which would ultimately serve as the enabling biotechnological platform for therapeutic gene delivery in vivo, enabling the development of tumor-targeted gene therapy vectors that could be administered systemically, which would then seek-out sites of cancerous histopathology and accumulate to high levels in primary tumors and in the remote, occult, and otherwise inaccessible lesions of cancer metastasis.…”

The ‘timely’ development of Rexin-G: First targeted injectable gene vector (Review)

“…Rexin-G, the world's first-targeted, injectable gene vector to be validated in the clinic, is administered by simple intravenous infusion (A). Distributed by the systemic circulation, the tumor-targeted nanoparticles seek out the biochemical hallmarks of tumor histopathology (11,12) and accumulate selectively in metastatic lesions that have spread throughout the body (see insert). Conceptually, Rexin-G is a small but complex gene delivery vehicle (3), which is based on the structural engineering of the retroviral core but is devoid of viral genes-carrying instead, a lethal form of the human cyclin-G1 gene as its molecular 'payload'.…”

“…The review assumes a posture of the medical avant-garde, but does not assume that the reader is either a molecular, genetic, or biotechnological cognoscente. The subject matter addressed herein is necessarily focused on very recent developments in the field, while pertinent background material on applied nanotechnology (2,3), gene delivery vehicles (4)(5)(6), cell cycle control (7,8), virotherapy (9,10) and tumor targeting technologies (11)(12)(13)(14)(15), as well as early clinical trials of Rexin-G (16)(17)(18) and associated histopathology (19) are available in the existing literature. The text is divided into discrete subsections in which critical distinctions are made and considered opinions are presented, in a strategic flow of contextual information that travels from the theoretical to the clinical to the analytical and beyond, to the evolving praxis of modern medicine.…”

“…The delivery of cytocidal genetic constructs that effectively destroy the target tumor cells and/or their associated blood supply eliminates the major problems associated with insertional mutagenesis and engraftment of genetically altered cells, which has plagued gene therapy applications in the past. The singular limitation of retroviral vectors that previously stymied the clinical development of these otherwise ideal gene delivery vehicles was the inability to effectively target these stealth nanoparticles under physiological conditions (22)(23)(24), a precondition that remained daunting until the advent of pathotropic targeting (11)(12)(13)(14)(15)(16)(17)(18)(19) (also see section 4).…”

Post-intervention vessel remodeling

“…Accordingly several biologic targeting systems have recently been introduced. [4][5][6][7][8][9][10] In spite of relatively low transfection efficiency adenoviruses are still the most widely used viral vectors for vascular applications since they can transfect both proliferating and non-proliferating cells. 2,11 However, adenoviruses also transfect many unwanted organs and peripheral blood monocytes.…”