Therapeutic RNA Development

RNA has emerged as a promising modality for various therapeutic applications, including RNA vaccines, CRISPR genome editing, and T-cell therapies including CAR-T. VectorBuilder provides a comprehensive end-to-end RNA platform to translate your RNA development from concept to clinic, supporting every stage from in vitro transcription (IVT) vector design through functional validation and into GMP manufacturing.

Highlights

Integrated Technical Expertise

Expert guidance in each stage of RNA and LNP therapeutic development to maximize performance across applications.

End-to-End Solutions

Streamlined therapeutic RNA development with one-stop IVT vector design, manufacturing, and functional validation services.

CRISPR Applications

Extensive experience in optimizing RNA design and LNP delivery for precise and highly efficient genome editing, both in vitro and in vivo.

CAR-T Development

Achieve rapid, transient, and robust CAR expression using IVT RNAs for both ex vivo and in vivo T-cell engineering.

VectorBuilder helps you design, optimize, validate,
and manufacture RNA therapeutics – all in one place, faster than ever.

Talk to our experts now!

Workflow for Therapeutic IVT RNA Development

IVT Vector
Design & Cloning

IVT RNA Production

LNP Encapsulation

Quality Control (QC)

Functional Validation

Optimal design of IVT RNA vectors is key to therapeutic RNA development. By applying Good Vector Practice (GVP) and our extensive RNA engineering expertise, we address important quality considerations such as expression, stability, and efficacy early in the design process, ensuring that each RNA therapeutic is primed for optimal performance from the outset.

We offer multiple modalities including mRNA, saRNA, circRNA, and other small RNAs for diverse applications. Our technical team can also help you choose appropriate IVT backbones and optimize key RNA components such as coding sequence, 5’/3’ untranslated region (UTR), polyA tail, and Kozak.

VectorBuilder has developed a suite of proprietary technologies and reagents to streamline IVT RNA manufacturing, with fully scalable platforms that accommodate projects of varying size and clinical need. To prioritize therapeutic potency from the earliest stages, we offer a range of capping methods, nucleotide modifications, and high-efficiency proprietary purification strategies that enhance in vivo expression while minimizing immunogenicity. Cell-free or animal-free production is also available for large-scale RNA manufacturing.

Click here to read more about our IVT RNA production service.

Lipid nanoparticles (LNPs) are crucial for the effectiveness of RNA therapeutics, as they maintain RNA stability and facilitate efficient cellular delivery. VectorBuilder excels in producing homogeneous LNPs with high encapsulation efficiency. Notably, we specialize in LNP surface engineering, such as optimizing LNP formulations and conjugating tissue-specific antibodies, to enhance the tissue specificity and efficacy of RNA therapeutics.

Click here to read more about our therapeutic LNP engineering service.

Delivering RNA therapeutics with consistent quality and performance relies on rigorous and well-designed QC measures. RNA integrity, sterility, and purity (e.g. endotoxin and dsRNA residuals) are among the most important quality attributes for preclinical and clinical applications. At VectorBuilder we offer comprehensive, fully customizable QC solutions for both IVT RNA and LNP-encapsulated RNA and plasmids to ensure the reliability, safety, and potency of your product.

To streamline your RNA development from idea to application, our experts can help you conduct functional validation studies to assess the efficiency, efficacy, and safety of your RNA therapeutics, both in vitro and in vivo, across various applications such as CRISPR, CAR expression, and cancer treatment. For in vivo validation, studies can be conducted in a range of animal models including rodents and non-human primates (NHPs).

CRISPR Genome Editing

CRISPR IVT RNA is becoming a popular approach for genome editing due to its many advantages, including promoter-independent editing across cell types, no risk of genomic integration, and transient expression that helps minimize off-target effects. In addition, Cas9 mRNA and gRNA can be co-encapsulated in LNPs, leading to more efficient gene delivery and editing both in vitro and in vivo when compared to traditional vector systems.

Find more about our collection of fully-validated research-grade premade CRISPR RNA products.

Codon-Optimized HiExpress™ RNA

With VectorBuilder’s extensive expertise in vector design and engineering, we have developed sequence-optimized HiExpress™ hSpCas9 and adenine base editor (ABE) RNA products that drastically increase target protein expression and genome editing efficiency.

HiExpress™ hSpCas9
HiExpress™ ABE

HiExpress™ hSpCas9 IVT mRNA developed by VectorBuilder has high expression and editing efficiency_2

Figure 1. HiExpress™ hSpCas9 IVT mRNA developed by VectorBuilder shows the highest gene expression and editing efficiency among human Cas9 variants. (A) Western blot analysis and (B) quantification of normalized Cas9 expression relative to hSpCas9 in HEK293T cells 24 hours post-transfection with 1 ug of either hSpCas9 IVT mRNA, HiExpress™ hSpCas9 IVT mRNA, or a non-treated control (NC). (C) T7E1 editing assay in HEK293T cells 24 hours post-transfection with 1 ug of gRNA and the indicated amounts of hSpCas9 or HiExpress™ hSpCas9 IVT mRNA. The blue asterisk indicates the intact PCR amplicon and the pink arrows indicate the fragments generated by T7E1. Therefore, less HiExpress™ RNA is required to achieve comparable editing efficiency in cells, making it ideally suited for gene editing in therapeutic and biotechnology applications.

Validation of Adenine Base Editor (ABE) mRNAs in vitro

Figure 2. VectorBuilder’s HiExpress™ ABE mRNAs show extraordinary base editing efficiency in vitro. mRNAs coding for (A) ABE8.20-m and (B) ABE7.10 were co-transfected with identical gRNAs to HEK293T cells. Genomic DNA was extracted 48 hr post-transfection, and the target region was amplified by PCR and subjected to NGS. A to G editing (asterisks) was detected within the editing windows (dashed box). The highest editing rates of 97.7% (A) and 95.6% (B) were achieved on the 5th nucleotide.

In Vivo Knockout

In vivo CRISPR IVT RNA knockout.

Figure 3. VectorBuilder’s CRISPR RNA solution for in vivo gene knockout. (A) Experimental timeline of CRISPR IVT RNA knockout in vivo. Mice were intravenously injected with 3.0 ug/g of Cas9 mRNA/gRNA mixture co-encapsulated in LNPs or PBS. On Day 7, liver genomic DNA was extracted, PCR amplified, and subjected to a T7E1 assay to confirm editing. (B) Gel electrophoresis of the T7E1 assay confirming editing (asterisked band). Each lane represents one biological replicate.

In Vitro Knockout

Single-gRNA
Dual-gRNA

Figure 4. CRISPR-mediated gene knockout in vitro with a single gRNA. (A) IVT Cas9 mRNA was transfected into HEK293T-EGFP cells with two types of EGFP-targeting gRNA. EGFP expression in non-treated (NC) and transfected cells was observed by microscopy (B) and quantified using flow cytometry (C, D). (E, F) The editing to EGFP genes on the genome was further confirmed by T7E1 assay and Sanger sequencing.

Validation_of_hSpCas9_mRNA_knockout_with_dual_gRNAs

Figure 5. CRISPR-mediated gene knockout in vitro with dual gRNAs. (A) A vector coding for Cas9 was linearized and subjected to in vitro transcription to produce Cas9 mRNA, which was then co-transfected with two gRNAs targeting the same gene into NIH/3T3 cells. (B) Detailed experimental design of the gRNAs targeting exons 2-4 of the gene of interest. Primer pairs P1 and P2 were designed for PCR validation to amplify the regions surrounding the target sites. (C) Gel electrophoresis of PCR amplified genomic DNA confirms gene editing.

CAR-T Therapy

CAR-T therapy utilizes genetically engineered T cells expressing a chimeric antigen receptor (CAR) to treat clinical complications such as cancer and autoimmune diseases. Increasingly, IVT RNA is being used to deliver and express CARs in T cells, both ex vivo and in vivo, due to its transient expression, ease of manufacturability, and reduced immunogenicity. Compared to certain viral vectors (e.g. lentivirus), RNA does not integrate into the target cell genome and therefore makes the resulting CAR-T cells only transiently active, minimizing the risks of insertional mutagenesis and long-term toxicity. Combined with RNA’s quicker production and lower cost, it is especially suitable for early-stage personalized preclinical testing. In addition, LNP encapsulation ensures efficient and targeted delivery of CAR constructs, making it increasingly attractive for in vivo CAR-T development. VectorBuilder’s comprehensive LNP-RNA platform accelerates your CAR development from concept to application by helping you design, optimize, and validate CAR constructs.

Find more about our collection of fully-validated research-grade premade CAR RNA products. Alternatively, you can explore CAR expression vectors in our highly intuitive Vector Design Studio.

mRNA UTR optimization validation

Figure 6. Human CAR-T cells generated using VectorBuilder’s LNP-mRNA exhibited cytotoxicity against CD19+ cells. (A) Primary human T cells were transfected with LNP encapsulated anti-CD19 CAR mRNA. The killing function of the CAR-T cells was validated by co-incubation with CD19+ Raji cells and measuring the released lactate dehydrogenase (LDH). (B) Two IVT mRNAs coding anti-CD19 CAR with different co-stimulatory domains, 4-1BB (CD19-BBz, 1949 nt) or CD28 (CD19-28z, 1931 nt), were encapsulated in LNPs. (C) Activated human T cells were transfected with the corresponding LNP-mRNA and validated for CD19 CAR expression. (D) T cell-induced cytotoxicity was measured by LDH assay 18 hours after co-incubation of CAR-T cells with Raji cells at various effector-to-target (E:T) ratios.

Incorporation of a miR-122 targeting site into the 3 UTR reduces liver specific expression of IVT mRNA

Figure 7. Optimized LNP formulation increases RNA transfection efficiency in primary human T cells. (A) Activated T cells were transfected with LNP-encapsulated EGFP mRNA at 6 ug mRNA per 1×10⁶ cells. (B) Denaturing agarose gel electrophoresis confirmed the correct length and integrity of the EGFP mRNA before encapsulation. (C) The particle size and Zeta potential of the LNPs were measured before T cell transfection. (D) EGFP expression was imaged and analyzed 24 hours post-transfection using fluorescence microscopy and flow cytometry.

Beyond CRISPR and CAR-T, we also support the development of mRNA vaccines
and T cell engagers for next-generation cancer therapies.

Talk to our experts now!

Resources

FAQ

Why use IVT RNA to deliver the CRISPR system?

CRISPR-Cas9 can be delivered through a variety of methods including plasmid, recombinant virus, gRNA-Cas9 RNP complex, and as a mixture of gRNA and Cas9 IVT mRNA. Each of these methods has its own advantages and limitations which allows researchers to select the method tailored to their application for maximum efficiency with minimal off-target and undesired effects, with IVT RNA rapidly emerging as one of the most promising approaches.

Plasmids are generally the easiest to generate in large quantities cheaply and can be delivered through chemical transfection or electroporation which also tends to be simple. However, due to their method of delivery, they are largely limited to in vitro applications. In addition, the efficiency of plasmid transfection can vary widely between cell types and expression is dependent on cell-type specific promoter activity, which can hinder their efficiency in certain systems. As a result, plasmids tend to use highly active promoters which can lead to high-expression levels of Cas9 which can increase off-target editing and random integration of plasmid DNA into the host genome.

Recombinant viruses tend to be another popular method for delivery of CRISPR components as they can be used in difficult-to-transfect cells and can be used to create stably-expressing Cas9 cell lines for multiple gRNA experiments. However, this system has many of the same limitations of plasmid delivery including cell-type dependent promoter specificity and elevated risk of off-target effects due to prolonged Cas9 expression. In addition, recombinant viruses pose an elevated risk of insertional mutagenesis, especially in retroviral and lentiviral systems where genomic integration of the Cas9 transgene occurs.

Delivery of a gRNA-Cas9 RNP complex circumvents many of the limitations of the plasmid and recombinant viral systems allowing for rapid editing as neither transcription nor translation needs to occur. However, this method is largely limited to in vitro applications as it requires electroporation for efficient delivery. Editing by this method occurs rapidly and quickly drops off as Cas9 is degraded in host cells and is therefore widely limited by the availability of Cas9 protein.

In contrast to the above methods, delivery of a mixture of gRNA and Cas9 IVT mRNA has rapidly emerged as one of the most efficient methods for genome editing with minimal off-target effects. gRNA and Cas9 IVT mRNA can be delivered together co-encapsulated in lipid nanoparticles or chemical transfection reagents making the system suitable for both in vitro and in vivo applications without risk of genomic integration. Further, no transcription is required, making expression independent of cell-type specific promoter activity and translation occurs relatively quickly, allowing for rapid and efficient editing. Compared to gRNA-Cas9 RNP complex, mRNA promotes more prolonged expression of Cas9 allowing for sustained levels of editing; however, the mRNA is eventually degraded and editing only occurs transiently, limiting off-target effects.

In summary, delivery of CRISPR-Cas9 components through plasmids, recombinant virus, and gRNA-Cas9 RNP complex methods have limitations that limit their use to a number of applications and in addition have downsides including limited efficiency, increased risk of off-target effects, and potential for insertional mutagenesis. Instead, delivery of a mixture of gRNA and Cas9 IVT mRNA has emerged as one of the most efficient methods of genome editing due to its numerous advantages and fewer limitations and should be considered for genome editing applications.

Should I use single gRNA or dual gRNAs for CRISPR-mediated knockout?

For CRISPR-mediated genome editing, Cas9 nuclease is directed to the target site of site-specific guide RNA (gRNA) in the genome to create DNA cleavage. In most cases, to generate simple gene knockout, a single gRNA can be used together with Cas9 to generate a double-strand break (DSB), which is then inefficiently repaired by the non-homologous end joining (NHEJ), resulting in permanent mutations, such as small insertions or deletions, at the site of repair. A subset of these mutations will result in loss of function of the gene of interest due to frameshifts, premature stop codons, etc.

Dual gRNAs can be used if Cas9_D10A nickase is being used to target the two opposite strands of a single target site. In this approach, the nickase enzyme will generate single strand cuts on both strands, one guided by each of the two gRNAs, resulting in DSBs at the target site. Generally, this method reduces off-target effects of CRISPR/Cas9 expression because targeting by both gRNAs is necessary for DSBs to be generated.

Dual gRNAs can also be used when Cas9_D10A nickase and an exogenous donor DNA template are being used to introduce specific base-changes (e.g. knockins) into a gene of interest. In this approach, the two opposite strands would be targeted by the two gRNAs at two sites flanking the desired mutation site, and homology-directed repair (HDR) pathways make use of the exogenous donor template to repair the excised sequence.

How does the RNA-based CAR-T solution compare to other viral and non-viral delivery systems?

A detailed comparison of LNP-mRNA, lentivirus, and piggyBac-based CAR-T delivery systems is summarized in the table below:

Feature	LNP-mRNA	Lentivirus	PiggyBac Transposon
Delivery route	Ex vivo or in vivo.	Usually ex vivo.	Ex vivo.
Cell specificity	Intrinsic LNP tropism is often liver; T-cell targeting requires formulation/ligands (e.g. antibody) for ex vivo and in vivo use.	Pseudotyping (e.g. VSV-G) gives broad tropism; ex vivo transduction of isolated T cells is routine and highly effective.	Delivered into T cells via electroporation, which shows high efficiency when optimized.
Expression time	Transient. Expression begins within hours and lasts for days up to ~1 week. Repeated dosing yields repeated expression.	Stable, long-term expression due to host genome integration; expression lasts for months to years (effectively permanent in dividing T cells).	Stable, long-term genomic integration when transposase mediates insertion; expression persists like viral integration unless excised.
Safety	No insertional mutagenesis risk.	Risk of insertional mutagenesis due to genomic integration, which raises concerns for clinical use.	Risk of insertional mutagenesis due to stable integration into TTAA sites, which raises concerns for clinical use.
Cargo capacity	LNPs can deliver 4-10 kb of large mRNAs; longer mRNAs show reduced stability and translation efficiency.	Up to 9.2 kb; larger constructs compromise titer and transduction efficiency.	Very large capacity; can accommodate up to ~30 kb of DNA.
Immunogenicity	No viral proteins are produced so overall lower adaptive anti-vector immunity. mRNA and LNPs can sometimes stimulate innate immunity; nucleoside modifications and optimized lipids reduce immunogenicity.	Viral proteins (especially residual from vector prep) and integrated transgene expression can elicit immune responses. Patients can develop anti-vector or anti-CAR immune responses.	No viral proteins are produced so overall lower adaptive anti-vector immunity. Delivery via electroporation avoids viral proteins but electroporation itself causes cell stress.
Dosage controllability	High controllability due to transient expression. Dosing can be titrated (i.e. dose and frequency), which enables repeated administrations. Good for short-term programs or on-demand CAR expression to limit toxicity.	Low controllability once integrated due to persistent expression. Extensive additional engineering (e.g. regulated promoters or safety switches) is necessary to systematically control CAR expression.	Low controllability once integrated due to persistent expression. Extensive additional engineering (e.g. regulated promoters or excisable transposons) is necessary to systematically control CAR expression.