Eliminate Protein and Antibiotic Markers

December 21, 2022 / by Jim Williams, Ph.D.

Advantages of the Nanoplasmid RNA-OUT Marker System

For decades, the same plasmid technology has been used in vaccination, cell and gene therapy, and as a raw material in viral vector and RNA production, but that technology often isn’t a good fit for the processes where it’s being used. Now, with the growing availability of Nanoplasmid™ vectors, that is changing.

Comparing plasmids
Canonical plasmids contain antibiotic resistance markers in bacterial backbones greater than 2000 bp. With Nanoplasmids, smaller backbones increase expression level and durability while reducing cell-transfection-associated toxicity and transgene silencing that can occur with canonical plasmids. Nanoplasmid vectors are a proven transformative replacement in a wide variety of applications, offering greater safety and efficiency than traditional plasmids.

In my previous post on Nanoplasmids, I discussed structured DNA repeats. In this second part of an ongoing series of posts, I delve deeper into the science of Nanoplasmids, with the focus of this discussion being antibiotic markers.

Antibiotic markers in cell and gene therapy products
Vector-encoded antibiotic markers can be transferred to environmental bacterial organisms through horizontal gene transfer (HGT). For example, HGT has been documented to occur through unintended release of plasmid vectors from laboratories or manufacturing facilities that then transform and propagate in freshwater environmental organisms.

HGT is a well-known safety issue; both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have recently recommended that antibiotics not be used in bacterial selection for gene therapy products. Further, certain types of antibiotic resistance markers, such as genes encoding β-lactams such as ampicillin resistance markers, are particularly problematic since these FDA and EMA guidances state that selection using β-lactams should not be used during manufacture of a therapeutic product due to hypersensitivity reactions to β-lactams in some patients.

Read more about Eliminating Antibiotic Resistance Gene Transfer Risks in Cell & Gene Therapies in our published White Paper.

Are protein markers any better?
The majority of antibiotic-free selection markers for plasmid maintenance in bacterial cells are protein-based systems, such as those designed for:

• Plasmid-borne complementation of an essential gene that has been deleted from the chromosome (auxotrophy complementation)
• Plasmid maintenance systems using post segregational killing

However, vector backbone encoded proteins in gene therapy products when expressed may interact with cellular components to alter cell functions or viability and will likely induce an immune response that may be detrimental to the patient.

This later issue is shared with antibiotic markers, and has been shown to be a concern with AAV vectors due to high frequency reverse packaging of the bacterial backbone during production. This results in significant bacterial backbone encoded antibiotic resistance marker packaging into AAV viruses, and subsequent marker gene transfer to a patient during therapy.

Eliminating protein and antibiotic markers using non-coding RNA markers
Ideally, bacterial selection would be mediated by a non-coding selection marker. These have been developed, as:

• Plasmid borne non-coding mRNA that functions by RNA/RNA interaction to inhibit translation of a target chromosomally expressed gene, or
• by Operator Repressor Titration wherein a plasmid contains several operator sequences that titrate the repressor, allowing expression of a repressor regulated selectable marker gene

One of these RNA/RNA interaction non-coding selection marker systems, the RNA-OUT marker system, has a high yield robust manufacturing process that has been scaled to the 200g lot scale in cGMP manufacture. RNA-OUT vectors have also been tested without safety issues in multiple clinical trials.

Nanoplasmid vectors use the RNA-OUT antibiotic-free marker and have additional intrinsic advantages, notably that 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, dramatically reducing the risk of HGT. Additionally, the Nanoplasmid backbone has no coding capacity, eliminating the risk of AAV virus mediated vector backbone encoded protein marker transfer to patients.

Critically, retrofits of existing Lentiviral, AAV or mRNA vaccine vectors will not change the sequence or composition of the vector payload, Lentivirus, AAV virus or mRNA transcription unit in these cases.

In my next post discussing the potential of Nanoplasmids, I’ll touch on vector backbone mediated innate immune activation and why this can lead to cell death, silenced transgene expression, and reduced product efficacy, showing a different perspective of the platform to improve cell and gene therapy performance.

• Want to learn more? Visit the Aldevron Nanoplasmids page
• Watch our on-demand webinar, Genome Editing Tools: Beyond Discovery
• Download our Nanoplasmid brochure
• Download a bibliography of Nanoplasmid resources
• Have a topic to suggest? Let us know