Structured DNA Repeats

October 12, 2022 / by Jim Williams, Ph.D.

A plasmid replication challenge

For decades, the cell and gene therapy industry has been using the same plasmid technology in vaccination, cell and gene therapy, and as a raw material in viral vector and RNA production. In some cases, that technology isn’t a good fit for the processes where it’s being used. Now, with the growing popularity of Nanoplasmid™ vectors, that is starting to change.

While canonical plasmids contain antibiotic resistance markers in bacterial backbones greater than 2000 bp, smaller backbones increase expression level and durability while reducing cell-transfection-associated toxicity and transgene silencing that can occur with canonical plasmids.

With a small backbone and lack of antibiotic markers, Nanoplasmid vectors have proven to be a transformative replacement in a wide variety of applications, offering greater safety and efficiency than traditional plasmids. In a series of posts, I’ll delve deeper into the science of Nanoplasmids, sharing thoughts on the technology and how it can improve cost-effectiveness in discovery and manufacturing processes.

Addressing yield and stability issues
Two of the leading biotechnology platforms, adeno-associated virus (AAV) gene therapy vectors and mRNA vaccines, encode structured DNA elements essential for their function. AAV transfer plasmids contain highly structured palindromic Inverted Terminal Repeats (ITRs) flanking the “payload” transcription unit. These ITR repeats, which contain the viral replication origin and viral packaging signals, are highly unstable and difficult to replicate in E. coli, creating challenges for AAV transfer plasmid manufacture.

mRNA vaccines are manufactured from poly(A) -repeat containing template plasmid vectors that contain an antigen gene (prefusion stabilized spike protein in the case of the approved Moderna or BioNtech COVID-19 mRNA vaccines) downstream of a bacterial promoter, and upstream of a poly(A) repeat.

To manufacture the mRNA vaccine, the template vector is produced in E. coli, then linearized by restriction digestion downstream of the poly(A) repeat. mRNA encoding the antigen gene with a stabilizing poly(A) tail is then produced by in vitro runoff transcription. Similar to ITR repeats, the long (80-120 bp is typical) poly(A) repeats are often prone to deletions and the vectors are often low yielding in E. coli.

Using E. coli strain engineering approaches
To stabilize structured DNA repeats in AAV and mRNA vectors, the typical approach is to use recombination deficient hosts such as SURE or Stbl4. While these strains can be used to stabilize and produce AAV and mRNA vectors in small scale shake flask culture, the multiple mutations characterizing these strains lead to very poor biomass and vector yields in the large-scale fermentation required for vector production for clinical and commercial applications.

As an example, Aldevron’s REVIVER host strain, an engineered cell line that improves stability and replication of structured DNA sequences while retaining high fermentation productivity, is a superior high yielding E. coli host for manufacture of AAV transfer plasmids and poly(A) repeat containing mRNA template plasmids.

Improved AAV transfer plasmid and mRNA template vector design
Fermentation yields are often lower with AAV transfer plasmid and poly(A) repeat containing mRNA template plasmids than standard plasmids. Poor manufacturing yields appears to be due to the plasmid backbone encoded pUC replication origin having difficulty replicating closely positioned structured DNA sequences to high yield. That’s where an alternative R6K replication origin-based vector backbone, the Nanoplasmid backbone, comes into play. It has improved fermentation manufacturing yields up to five times higher with structured DNA containing vectors.

Nanoplasmid vectors have additional intrinsic advantages, notably they do not include antibiotic resistance markers. This is safer than plasmids that rely on antibiotic resistance for bacterial selection. The vectors also contain a specialized bacterial R6K replication origin in place of the traditional pUC replication origin, making these vectors replication-incompatible with native organisms.

This is an additional safety factor since Nanoplasmids can only replicate within the specialized E. coli host strain used for manufacturing. Critically, retrofits of existing AAV transfer vectors or mRNA vaccine vectors will not change the sequence or composition of the vector payload, AAV virus or mRNA transcription unit in these cases.

In my next post discussing the potential of Nanoplasmids, I’ll touch on antibiotic markers and why they can be a regulatory nightmare, showing a different perspective of the platform in general along with the new backbones for AAV and mRNA vectors.

• Want to learn more? Visit the Aldevron Nanoplasmids page
• Download our Nanoplasmid brochure
• Download a bibliography of Nanoplasmid resources
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