New system for therapeutic gene delivery increases transgene expression up to 100-fold

Advanced engineering of a mini-intronic plasmid (MIP) system designed to carry a therapeutic gene can significantly enhance the expression of the transgene delivered using an adeno-associated viral (AAV) vector. The ability to increase transgene expression by up to 40 to 100-fold, which would reduce the cost of manufacturing and perhaps also lessen the immune response of AAV/MIP-based gene therapy, is reported in Human Gene Therapy, a peer-reviewed journal from Mary Ann Liebert, Inc., publishers

The researchers discuss the potential implications of enhanced transgene expression on the doses needed to achieve a therapeutic response and the flexibility the small intronic sequences offer, allowing them to be used in both DNA plasmids and viral delivery vectors.

“Careful observation of the expression characteristics of different vector designs sometimes leads to unexpected findings,” says Editor-in-Chief Terence R. Flotte, MD, Celia and Isaac Haidak Professor of Medical Education and Dean, Provost, and Executive Deputy Chancellor, University of Massachusetts Medical School, Worcester, MA. “In this case, the authors found that a very substantial increase in the amount of transgene expression (up to 100-fold) could be achieved from rAAV vectors by including essential bacterial plasmid elements in an upstream intron. This could present substantial advantages for future in vivo gene therapy,”

Recombinant AAV vectors are showing promise in clinical applications, albeit the ability to enhance gene expression would potentially reduce the dose required for many indications. The fact that our engineered intron can enhance rAAV-mediated transgene expression by up to 40- to 100-fold is remarkable. The ability to reach the same level of transgene expression, with a 40- to 100-fold lower dose would reduce the cost of manufacturing and perhaps reduce the immune response observed after systemic administration of the vector in humans. Moreover, these small intronic sequences seem to work in both DNA plasmids and viral vectors providing a universal addition that may enhance transgene expression from any vector.

Human Gene Therapy – A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo


We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.


The basic gene expression unit contains a promoter that initiates transcription of a particular gene, a gene coding region, and a polyadenylation signal that correctly terminates the transcription. This basic gene expression unit has become the default structure for most commonly used gene expression cassettes in both biomedical research and clinical areas. Each potential component of the expression cassette provides different abilities to facilitate robust transgene expression.

In quiescent tissues, minicircle DNA vectors devoid of the bacterial backbone can provide 10 or more times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. The reason for this is that transcriptional silencing of the transgene occurs when >1 kb of spacer DNA exists between the 5′ end of promoter and 3′ end of polyadenylation (polyA) site. Based on these findings, we constructed a mini-intronic plasmid (MIP) in which the components required for plasmid propagation in bacteria (bacterial replication origin and selectable marker) are positioned within an intron of a noncoding exon contained within the expression cassette. Because there is virtually no spacer outside of the expression sequence, MIP vectors are not subject to transgene silencing and in addition provide 2–10 times or higher levels of transgene expression compared with minicircle vectors in vivo and in vitro.

The detailed mechanism of MIP-generated enhanced expression over minicircle DNAs and plasmid DNAs is not clear. It has been previously reported that introns may increase gene expression through enhancing mRNA export from the nucleus to the cytoplasm. Splicing of promoter proximal introns has been shown to provide enhanced transgene expression. Additionally many recombinant genes are expressed inefficiently when their introns are removed.

With the success of MIP vector, we then tested the potential of MIP intron (bacterial replication origin and selectable marker sequences) in promoting transgene expression from adeno-associated virus (AAV) vectors. Enhancing the expression abilities of AAV vectors will significantly lower the dose of the vector needed to achieve therapeutic levels of expression, reducing the cost and potential for vector mediated immune responses directed against the vectors’ capsids in transduced cells

SOURCES – Eurekalert, Human Gene Therapy