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September 18, 2024 by Tyler Kozisek

Increasing expression AND improving yields

One of the main limitations of nonviral gene delivery is that transgene expression is generally very low compared to viral based gene delivery methods. In general, the low expression is attributed to promoter inactivation by immunostimulatory motifs in the bacterial region of plasmids (i.e., region containing bacterial origin of replication and selectable marker).

Removal of the bacterial region has been shown to increase transgene expression (e.g., minicircle vectors) compared to traditional plasmids, however, these bacterial region free vectors are not replication competent, meaning production and scale-up of minicircle vectors is difficult and costly.

Alternatively, the Nanoplasmid^TM platform can offset shortcomings of both traditional plasmids (low transgene expression) and minicircle vectors (low production yields and costly scale-up) by incorporating a short bacterial region (<500 bp) that has no coding capacity, uses antibiotic-free selection, is host strain restricted, and has been engineered to remove all non-essential sequences.

Nanoplasmid’s minimized bacterial region can dramatically increase transfection efficiency and transgene expression in a variety of cell types and gene delivery applications. Not sure if Nanoplasmid will help in your nonviral gene delivery application? Aldevron offers eight different reporters: Cytomegalovirus (CMV), CAG, Elongation factor-1 alpha (EF1α)(intron), and EF1s with either enhanced green fluorescent protein (EGFP) or firefly luciferase. Not sure on which flavor to test? Let me summarize some of the pros and cons to help you make that decision.

CMV Reporters
CMV reporters drive high transgene expression in many different mammalian cell lines, primary cells, and in vivo due to the CMV being a strong viral promoter. The Nanoplasmid CMV reporters have the potent human T-lymphotropic virus type 1 (HTLV-I) R region 5’ untranslated region (UTR) transient expression enhancer incorporated as part of exon 1 and intron 1 downstream of the CMV promoter. The mechanism of enhancement from the HTLV-I R in the CMV reporters is increased mRNA translation efficiency. That is, more transgene is produced per transgenic mRNA rather than increasing overall cytoplasmic mRNA.

CAG Reporters
The CAG promoter used in the Nanoplasmid off the shelf reporters is a strong hybrid promoter consisting of the CMV early enhancer (C), the first exon and intron of the chicken beta-actin gene promoter (A), and a splice acceptor from the rabbit beta-globin gene (G). The CAG promoter also drives high transgene expression levels in mammalian cell lines, primary cells, and in vivo, with the added benefit of long-term expression of transgenes during stem cell differentiation and less promoter silencing compared to a CMV promoter.

EF1α Reporters
The human EF1α promoter used in the Nanoplasmid off-the-shelf reporters is a constitutive promoter of human origin that has strong promoter activity in various cell types, especially in T cells. The Ef1α (intron) Nanoplasmid reporters have the EF1α core promoter along with the first intron of the human EF1α promoter.

The inclusion of the first intron of the human EF1α promoter has been shown to be key to achieving high levels of transgene expression. However, removal of the EF1α first intron to create a shorter Ef1α promoter (EF1S) can also achieve high levels of transgene expression.

Polyadenylation Signal
A modified bovine growth hormone (BGH) polyadenylation (poly(A)) signal is used on all off the shelf reporters. The BGH poly(a) signal was modified to remove any CpG motifs that can cause transgene silencing and vector inactivation due to recognition of unmethylated CpGs by transfected cells and activation of innate immune signaling pathways.

The Takeaway
The Nanoplasmid platform is a next generation plasmid bacterial backbone that can increase transgene expression, decrease innate immune activation and transfection toxicity, and reduce vector inactivation compared to traditional plasmids. Furthermore, Nanoplasmids make use of a strain restricted origin of replication and an antibiotic free selection system, which lessens regulatory concerns.

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Still on the fence on if the Nanoplasmid is right for your nonviral gene delivery application? Try one of Aldevron’s free off the shelf Nanoplasmid reporters, offered with EGFP or firefly luciferase to meet your in-house assay needs.

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