Gene therapy is rapidly growing field that utilizes viral and non-viral particles to deliver DNA or RNA to a patient’s target cells to treat intractable diseases, such as cancer and genetic diseases. The manufacturing of such particles is complex, potentially requiring months and resulting in heterogenous products with low yields of high-quality particles. Measurements of particle identity, purity, potency, safety, and stability are needed to ensure gene delivery system quality. NIST is developing approaches to provide advanced characterization of these critical quality attributes and of physical and infectious titer. NIST is also collaborating with federal and industry partners on developing and characterizing reference materials to make available to the public. These measurement tools and reference materials can help increase product understanding and accelerate gene delivery system development.
Viral vectors are a critical tool for delivering DNA or RNA into patient’s cells. Common viral vectors for gene therapy include adenovirus, lentivirus, and adenoassociated virus. Administration of viral vectors can occur in vivo or ex vivo, such as for adenoassociated virus or lentivirus CAR-T therapy, respectively. Heterogeneous production of viral vectors results in the presence of full, empty, or partially filled particles with therapeutic DNA or RNA. These varying states have immunogenic and functional impacts on a viral vector preparation. NIST is developing physical titer methods to quantitatively measure loaded viral particle state to provide manufacturers quality attributes on their viral vector preparation. Additionally, NIST is working on developing advanced measurements by imaging and flow cytometry of infectious titer to correlate with physical titer measurements. These physical and infectious titer measurements can provide holistic characterization of viral vector heterogeneity to advance understanding of viral vector quality.
Flow cytometry has traditionally been used to analyze cells, measure biomarkers or inner cellular molecules, or infected cells. Analyzing smaller entities, such as viruses, has shown to be extremely challenging; however, flow cytometry has become a powerful tool for their characterization because of the recent advancement in instrumentation and labeling reagent. The NIST Flow Cytometry Lab is utilizing the unique knowledge of staff and comprehensive collection of flow cytometers to conduct cutting edge measurements of viral vectors of ≥20 nm in size. Physical titer, viral packaging information, and potential contamination can all be quantitatively measured.
The goal of this project is to develop novel high-throughput light scattering microscopy to individually measure the mass of viral and nonviral particles to help elucidate particle loading, as well as size and concentration measurements, to create a robust and quantitative set of quality attributes for physical titer measurements.
Infectious titer is a critical measurement of viral vector function and potency. Traditional methods for infectious titer quantification include tissue culture infectious dose (TCID50) assays, plaque assays, and gene transfer assays as measured PCR and flow cytometry. NIST is developing protocols and improved measurement techniques which can reduce the variability and improve the reproducibility of infectious titer assays. Projects include automated plaque identification, lentivirus transduction, and pseudovirus infection and serum neutralization. Measurement methods include endpoint microscopy, time lapse imaging, and flow cytometry. High speed microscopy methods are under development to accelerate infectivity assay imaging.
See here for more information on pseudovirus and serology testing.
Fast and robust viral vector characterization methods are significantly needed for the quality assurance of viral vector production. Traditional viral titer methods are often based on certain assumptions, such as 2000 molecules of p24 in each lentiviral particle. NIST is developing protocols and improved measurement techniques for viral vector genomic assays. These assays can reduce the sample handling time and variability and improve the reproducibility of viral vector titration.
NIST is also developing techniques and protocols to evaluate the viral vector genome integrity. The genome integrity was assessed by RT-ddPCR (such as LV) or ddPCR (such as AAV) assays targeted to distant regions of the LV genome as shown.
Other genomic assays for characterizing viral vectors:
Reference materials can serve an important role is the development and commercialization of viral vector-based gene therapies. These materials can enable comparability of different viral vector preparations for physical and infectious titer measurements. NIST is collaborating with FDA and industry partners to characterize the next generation of viral vector reference materials. NIST is currently working on three reference material projects:
The copy number of integrated transgenes into target cell genomes is critical for assessing the safety and efficacy of engineered cellular therapeutic products such as CAR-T cells. NIST is evaluating whether vector copy number cells (VCN) can be used to benchmark integrated lentivirus copy number measurements in target cells. These VCN cells also serve as a basis for developing cell-based (Jurkat) reference materials that can be distributed between laboratories to validate bioprocesses associated with the manufacturing and evaluation of cell and gene therapies.
CAR-T VCN cell lines: NIST is working on the development of anti-CD19 CAR-T model cell lines by transducing Jurkat (Clone E6-1, ATCC) cells with a lentivirus expressing an anti-CD19 cassette and eGFP. The single cell clones of transduced Jurkat cell lines were selected, expanded, and validated by digital PCR to produce stable cell lines with N=1, N=2, N=3, and N=4 copies of the integrated provirus. The protein expression of anti-CD19 antibody and eGFP are being evaluated, and will be followed by functional study such as CAR-T killing assays. To learn more about this project, please contact Zhiyong He (zhiyong.he [at] nist.gov), Linhua Tian (linhua.tian [at] nist.gov), Lili Wang (lili.wang [at] nist.gov), or Hua-Jun He (hua-jun.he [at] nist.gov).
Extracellular vesicles (EVs) are nanometer-sized vesicles that contain bioactive lipids, RNAs and proteins, which can be transferred to recipient cells. EVs are important for physiological as well as pathological processes, such as coagulation and immune homeostasis, aiding cancer metastasis and spread of infectious diseases. These unique properties have spurred tremendous interest in the broad application of EVs in healthcare as potential disease biomarkers, regenerative medicines and therapies, and as drug delivery vehicles. Various characterization methods based on different principles have been developed for EV investigations, but their relatively small sizes and complex population and subpopulation heterogeneity, and lack of reference and/or control materials for purification and characterization pose key challenges to the successful translation of EVs to the clinic and beyond. The robust characterization methods identified in the EV work are applicable to other non-viral vectors including lipid nanoparticles (LNPs).
Research has been focused in four areas to develop some measurement assurance around EV technology:
Emerging Project:
NIST is looking for postdoctoral applicants interested in developing and applying novel characterization approaches to measure gene delivery system quality. Two-year fellowships are available through the NIST-NRC Postdoctoral Fellowship program. The program is open to US Citizens only, offers competitive salary and benefits, with research proposal and application deadlines annually on February 1 and August 1. Contact NIST staff and see the NIST-NRC webpage for more information.