Nanoplasmid® FAQs

A Next-Generation Plasmid Vector System

Read the FAQs on our next-generation plasmid vector system designed for cell and gene therapy applications. While answering the questions below, we cover reported therapeutic applications, their respective cell and gene editing modalities using Nanoplasmid, and any relevant resources and articles.

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How is Nanoplasmid different from other DNA technologies on the market?

Nanoplasmid encompasses all the performance benefits (i.e. improved transfection efficiency, reduced cellular toxicity after transfection, and improved transgene expression) of minicircles without the problematic low-yield manufacturing and purity complications from long and complicated processes. To learn more about Nanoplasmid’s performance benefits, please download our whitepaper: Next Generation Plasmid Technology Improving Performance Safety and Manufacturing for Today’s Therapies.  

Furthermore, Nanoplasmid’s antibiotic-free selection system is R6K strain-dependent, eliminating the chance of antibiotic resistance marker transfer to patients while reducing horizontal gene transfer seen in promiscuous pUC vector systems. If you would like to learn more about Nanoplasmid’s antibiotic-free selections system and how it can reduce your organization’s regulatory concerns, please read our whitepaper titled: Eliminated Antibiotic Resistance Gene Transfer Risks in Cell & Gene Therapies 

When comparing Nanoplasmid to synthetic linear DNA, it has superior performance in transfection efficiency, and the manufacturing of synthetic DNA is also limited by its in vitro transcription error rate (generally around 100-1000 increased mutation rate) as compared to traditional plasmid propagation, which includes repair mechanisms (mismatch repair) that maintain high fidelity.  

I already have an existing construct. Can I still use Nanoplasmid?
Yes, we will retrofit your construct into our Nanoplasmid vector system and support all your manufacturing needs, from research-grade to GMP-Source® to GMP materials. 
How does Nanoplasmid’s antibiotic-free selection system work?
The RNA-OUT platform works by repressing the expression of a counter-selectable marker (SacB) from the host chromosome. SacB encodes for the levansucrase enzyme, which creates a toxic environment in the presence of sucrose, leading to cell death. Transforming the antibiotic-free Nanoplasmid in the host expresses a 150bp RNA-OUT antisense RNA (ROUT) that binds the SacB mRNA, represses levansucrase expression, prevents toxicity, and maintains cell survival.  
Are there additional benefits of the R6K origin besides reduced horizontal gene transfer potential?
Nanoplasmid’s R6K strain dependence improves the safety profile by reducing the horizontal gene transfer potential compared to traditional and promiscuous pUC plasmids. Moreover, the proprietary REVIVER® host strain replicates structural DNA found in ITR-bearing plasmids used for AAV production and Poly(A) tails found in mRNA templates with higher integrity than typically comparable strains used to propagate pUC plasmids.
Can you share any data regarding transfection efficiency improvement using Nanoplasmid?

Many peer-reviewed articles and clients report lower cell death and improved transgene expression after transfection when using Nanoplasmid. 

You can see our full, up-to-date list of featured publications by downloading our bibliography here. 

How does Nanoplasmid improve transfection efficiencies?
Nanoplasmid improves transfection efficiencies through efficient and purposeful engineering of various elements. Its small backbone (<500 bp), lack of antibiotic selection systems and removal of bacterial coding regions offer unparalleled advantages over competing technologies. We believe the bacterial coding regions activate innate immune responses due to the generation of pathogen-associated molecular patterns or danger-associated molecular patterns, which often result in cell death after intracellular expression of materials of bacterial origin after transfection. 
What is the maximum/largest insert Nanoplasmid has expressed?
We have had partners and scientists insert genes of interest around the 20kb mark, and we have yet to find the upper limit of its capacity. The largest GOI currently in clinical trials is approximately 10kb. 
How is Nanoplasmid different from a minicircle?
Nanoplasmid has a small backbone (<500 bp), similar to the size of minicircles (100 bp), but it is manufactured in a proprietary host strain and batch-fed process without recombinase processing, which is what makes minicircles expensive to manufacture and impractical at scale.  
Has Nanoplasmid been used in any clinical trials?
The Nanoplasmid vector has been employed in at least 13 clinical trials comprised of eight completed phase I trials, two recruiting or ongoing phase I trials, one ongoing phase II trial, and two completed phase II trials. If you would like to learn more about the clinical applications of Nanoplasmid, please read our review published in Molecular Therapy-Nucleic Acids here. 
Has Nanoplasmid faced any regulatory issues from the Food and Drug Administration or the European Medicines Agency?
No, Nanoplasmid has not encountered regulatory issues from the FDA or EMA. Its antibiotic-free selection system and R6K strain eliminate the chances of antibiotic resistance marker transfer to the patient, fulfilling a recommendation from both organizations, and its R6K strain significantly reduces horizontal gene transfer within patients or the environment.
Can you control the copy number with Nanoplasmid?
We can control the copy number by using a temperature shift during the proprietary and patented manufacturing process. 
Has Nanoplasmid been used as a CRISPR HDR donor template?
Yes, with several different publications, a group from Genentech has reported using Nanoplasmid as a donor template for the manufacturing of CAR-T cells, as compared to linear double-stranded DNA (dsDNA) and a traditional pUC plasmid. They found that Nanoplasmid showed two and three-times fold edited CAR-T cells compared to a pUC plasmid and linear dsDNA, respectively. (1) an additional report was published in Nature by the Editas Medicines group, where they developed a novel high-efficiency knock-in method through negative selection of essential genes. (2) Another group at Stanford also developed a 14-day process for CAR-T cell manufacturing using a unique selection system they developed. (3) For a complete list of all the publications and reports using Nanoplasmid as a CRISPR HDR donor template, please view our Nanoplasmid Bibliography. 
Has Nanoplasmid been used in any Transposon applications?
Yes, Nanoplasmid has been used as the template for transposon therapy but rarely as the delivery vector for transposase expression. However, this is another possible application. Most groups we work with want the transposase enzyme for a relatively short duration. Therefore, the mRNA approach is often preferred to express the transposase, which Aldevron can also support. POSEIDA Therapeutics reported improved manufacturing, functionality/ phenotype of their CAR-T cells in a small cohort of their BCMA trial when using Nanoplasmid as their template compared to a conventional plasmid template. Additionally, Nanoplasmid has been used with several transposon systems, including piggyBac®, Sleeping Beauty, and TcBusterTM systems. For a complete list of all the publications and reports using Nanoplasmid in a transposon system, please view our Nanoplasmid Bibliography.  
How does Nanoplasmid improve the production of plasmid constructs used for AAV and mRNA manufacturing?

AAV: Nanoplasmid’s proprietary host strain REVIVER® has been demonstrated to produce higher plasmid yields (~3-8 times) than a typical Stbl3 strain while maintaining or improving ITR integrity in plasmids used for AAV. Furthermore, REVIVER® is a non-mucoid culture that allows for improved downstream processing of the final product.  

mRNA: Regarding mRNA template manufacturing, similarly to AAV, the REVIVER® host strain propagates constructs containing poly(A) tails while improving yield and maintaining or improving the structural DNA integrity.  

How can I evaluate Nanoplasmid in my research?

Please visit our nanoplasmid page to initiate your inquiry. An Aldevron colleague will reach out to gather additional details such as the number of constructs, quantities of each construct, timelines, etc. S/he will provide additional information about the pricing and connect you with our scientific staff to start the design phase.  

Aldevron respects, and does not ask for, sensitive information regarding your gene of interest (GOI) or additional details other than what is necessary for retrofitting/subcloning into the Nanoplasmid backbone. Generally, you should include two weeks for cloning and two weeks for production, not including the time required for gene synthesis as needed. Many partners request Aldevron’s standard Confidentiality Disclosure Agreement (CDA) before sharing details of their experiments. The Nanoplasmid constructs are shipped under a Limited Use Label License (LULL). 

After a successful evaluation of Nanoplasmid at the research level, how can I proceed to clinical trials?

For clinical trials for indications in human or animal health, you may require GMP (or equivalent) quality-grade plasmid DNA (pDNA). We will connect you to the Aldevron sales team in your geographic area to evaluate your needs, such as quality level, quantity, timeline, price, and legal/regulatory documentation and support. With 25+ years of experience, you can rest assured that all your questions will be addressed.  
 

Prior to initiating clinical trials, you will also require a clinical and commercial use license. We encourage you to reach out for a detailed discussion. While each license agreement is structured differently to accommodate your needs and requirements, a few common license elements include an upfront fee, maintenance fee, and success-based milestones. 

How should I proceed if my company wishes to in-source production of Nanoplasmid or engage a third-party contract manufacturer?
Aldevron uses patented and proprietary (a) bacterial cell lines, and (b) manufacturing process, and prefers to meet all your needs regarding Nanoplasmid manufacturing at all quality levels. In rare instances, Aldevron would agree to tech-transfer its proprietary technology to a partner or third-party contract manufacturer. Please reach out to discuss additional details.

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