Development of Cell Penetrating Peptides for Effective Delivery of Recombinant Factors into Target Cells.

The cell membrane is a protective layer that strictly controls the passage of molecules restricting the delivery of biomolecules such as drugs, oligonucleotides, peptides, and siRNA into the cells. This shortcoming has been overcome by the discovery of Cell-Penetrating Peptides (CPPs) that has undergone 30 years of evolution. To date, CPPs are largely modified to improve its efficacy and to suit the different delivery applications. The modes of CPPs penetration are still an unresolved mystery and requires further investigations to increase its effectiveness and to diversify its use. Despite having huge potential as a biomolecule carrier, CPPs also have some drawbacks. In this review, the natural and synthetic CPPs, the modifications that have been conducted on CPPs to improve its efficacy, its extended applications, modes of penetration and limitation as well as challenges will be discussed.

Transient Expression of Green Fluorescent Protein in Integrase-Defective Lentiviral Vector-Transduced 293T Cell Line.

Non-integrating lentiviral vectors or also known as integrase-defective lentiviral (IDLV) hold a great promise for gene therapy application. They retain high transduction efficiency for efficient gene transfer in various cell types both in vitro and in vivo. IDLV is produced via a combined mutations introduced on the HIV-based lentiviral to disable their integration potency. Therefore, IDLV is considered safer than the wild-type integrase-proficient lentiviral vector as they could avoid the potential insertional mutagenesis associated with the nonspecific integration of transgene into target cell genome afforded by the wild-type vectors.Here we describe the system of IDLV which is produced through mutation in the integrase enzymes at the position of D64 located within the catalytic core domain. The efficiency of the IDLV in expressing the enhanced green fluorescent protein (GFP) reporter gene in transduced human monocyte (U937) cell lines was investigated. Expression of the transgene was driven by the spleen focus-forming virus (SFFV) LTRs. Transduction efficiency was studied using both the IDLV (ID-SFFV-GFP) and their wild-type counterparts (integrase-proficient SFFV-GFP). GFP expression was analyzed by fluorescence microscope and FACS analysis.Based on the results, the number of the GFP-positive cells in ID-SFFV-GFP-transduced U937 cells decreased rapidly over time. The percentage of GFP-positive cells decreased from ~50 % to almost 0, up to 10 days post-transduction. In wild-type SFFV-GFP-transduced cells, GFP expression is remained consistently at about 100 %. These data confirmed that the transgene expression in the ID-SFFV-GFP-transduced cells is transient in dividing cells. The lack of an origin of replication due to mutation of integrase enzymes in the ID-SFFV-GFP virus vector has caused the progressive loss of the GFP expression in dividing cells.Integrase-defective lentivirus will be a suitable choice for safer clinical applications. It preserves the advantages of the wild-type lentiviral vectors but with the benefit of transgene expression without stable integration into host genome, therefore reducing the potential risk of insertional mutagenesis.