Improving Transduction Efficiencies

The number of cells transduced, or infected, by retroviral vectors is often insufficient to produce a therapeutic effect. Murine leukemia virus vectors require breakdown of the nuclear membrane during cell division for entry

Fig. 2 A. The retroviral genome is composed of three genes, gag, pol, and env, flanked by long terminal repeats (LTR) which contain sequences necessary for initiation and termination of transcription as well as signals for integration into chromosomal DNA. The packing signal, C, is required for packing of the retroviral genome into virions. B. Production of retroviral vector particles is achieved by cotransfection of three plasmids and expression of the viral proteins in trans. The transfer vector is the only sequence packaged into the vector particle as the packaging signal has been removed from the other constructs. Safety of the transfer vector has been improved by deletion of viral enhancer and promoter sequences from the 3'LTR, creating a self-inactivating (SIN) vector with no active viral promoters.

Fig. 2 A. The retroviral genome is composed of three genes, gag, pol, and env, flanked by long terminal repeats (LTR) which contain sequences necessary for initiation and termination of transcription as well as signals for integration into chromosomal DNA. The packing signal, C, is required for packing of the retroviral genome into virions. B. Production of retroviral vector particles is achieved by cotransfection of three plasmids and expression of the viral proteins in trans. The transfer vector is the only sequence packaged into the vector particle as the packaging signal has been removed from the other constructs. Safety of the transfer vector has been improved by deletion of viral enhancer and promoter sequences from the 3'LTR, creating a self-inactivating (SIN) vector with no active viral promoters.

into the nucleus and integration into the genome, thus limiting the number of cells that can be transduced. The traditional approach for enhancement of MLV vector transduction efficiencies has been stimulation of cell proliferation by the addition of various growth factors ex vivo.[4] Alternatively, lentiviruses, a subclass of retro-viruses, possess nucleophilic signals which permit entry into the nuclei of nondividing cells. However, lentiviral-based vectors are not yet widely used in clinical trials.

The entry of a retrovirus into a cell is also largely determined by binding of the envelope glycoprotein to its cellular receptor, and the distribution of the receptor on various cell types will influence the vector's host range. Envelope proteins from heterologous retroviruses can be used to create pseudotyped vectors with broadened host ranges. For example, pseudotyping with the VSV-G rhabdovirus envelope protein is a common technique used to improve stability and extend the host range of retroviral vectors.[5]

Although pseudotyping and stimulation of cell proliferation have been successful in improving transduction efficiencies of particular cell types ex vivo, such expansion of viral host range may not provide the cell specificity required for gene delivery in vivo. To improve transduction efficiencies in vivo, several methods involving modification of natural retroviral envelopes have been explored to target vectors to a specific cell type (Fig. 3). The retroviral Env protein is composed of two subunits, the surface (SU) protein containing the receptor recognition domain and the transmembrane (TM) protein which anchors the complex within the viral lipid envelope. Binding of SU to its receptor is thought to trigger conformational changes which result in exposure of a fusion peptide on the TM subunit and subsequent fusion of viral and cellular membranes. Most attempts to reengineer the receptor binding site of the SU subunit have involved replacement of the natural receptor-binding domain with a ligand or single-chain antibody with tropism for an alternative cell surface molecule and have resulted in a lack of virus-cell fusion.[6] Some strategies have resulted in limited success, such as the replacement of the MLV Env receptor-binding surface of SU with the peptide ligand, SDF-1a, which resulted in transduction of human cells via the SDF-1a receptor, CXCR4.[7] However, transduction efficiencies were low.

A second approach to Env targeting, called tethering, concentrates vectors on the surface of specific cell types by the addition of a second binding moiety to the natural Env protein. The native receptor binding domain remains functional and mediates virus-cell fusion. The insertion of a von Willebrand factor-derived collagen-binding sequence at the N-terminus of the MLV envelope has resulted in improved gene delivery in vivo to sites of exposed extracellular matrix within tumor vasculature following systemic administration in mice.[8]

An alternate approach, the use of adaptor proteins which function as a bridge between the native MLV Env and cell surface receptors, has generally failed due to a lack of virus-cell fusion. However, greater success has been observed with the avian leukosis virus (ALV) Env protein. Use of an adaptor protein consisting of the single-chain antibody, MR1, which binds to a tumor-specific form of the EGF receptor, joined to the extracellular domain of the natural ALV receptor resulted in successful

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment