Step 4

Summary

    There exists a wide variety of vectors used in gene therapy and genetic modification.  These extant vectors have particular strengths and weaknesses for various applications.  The current classification of existing vector systems for delivery of nucleic acid rests upon the traditional division between nonviral and viral vectors.  While nonviral vectors such as liposomes are limited in efficiency, viral vectors can provoke hazardous immune reactions.  Often, viral vectors are made incapable of replication by the removal of critical components used in forming functional virus particles.  Nonviral vectors are artificially engineered to gain the ability to deliver DNA.

    Exosomes, defined as 40-100nm membrane vesicles arising from the intralumenal vesicles of multivesicular bodies upon their fusion with the plasma membrane, have been found to contain diverse sets of mRNAs, miRNAs, and proteins as part of their involvement in intercellular communication.  Although a liposome is typically a synthetic phospholipid bilayer vesicle designed for encapsulating nucleic acid, exosomes have been recently categorized as a form of biological liposome.  A model initially described as the Trojan exosome hypothesis regarding the essentially borrowed use of the nonviral exosome pathway by viruses found experimental support in later studies.  Also, recent work has indicated the presence of mtDNA in exosomes released by astrocytes and glioblastoma cells.  These recent developments indicate that exosomes may constitute a new form of vector potentially suitable for genetic modification and gene therapy, given their natural tendency to transfer materials between cells in multicellular systems.

    At present, the movement of RNAs and proteins between cells via exosomes is a phenomenon that has been studied but not yet precisely controlled. Exosomal transfer of RNAs and proteins between cells has been documented in mast cells and neurons, cell types particularly noted for well-developed intercellular communication.  While the possibility of using exosomes to deliver specific DNA or RNA to cells has been raised in the literature, no particular method has yet been articulated for targeting genetic constructs to exosomes or using exosomes in a regulated manner to promote the spread of a genetic construct through a multicellular system.  As another barrier to the development of this system, the human mast cell line HMC-1, which has been used to characterize exosomal transfer, is traditionally cultured in media formulations based off of Iscove's Modified Dulbecco's Medium (IMDM) with added fetal bovine serum.  Ultracentrifugation is sometimes used with the intent of removing exosomes from serum.  Considering that sources of sera and centrifugation equipment and protocols may vary from laboratory to laboratory, creating a precise serum-free media formulation for the culturing of HMC-1 cells would be answering a major question in the field of exosomal research.  A leading extant formulation of serum-free medium, StemPro®-34 SFM Complete Medium, is designed for use in hematopoietic stem cells. Its use has been explored in primary cultures of human mast cells.  As the IMDM/FBS based medium formulation is standard for culturing HMC-1 cells, developing a serum-free media formulation for HMC-1 cells addresses a significant question in research towards developing the exosomal vector.