DNA research has come a long way since these two milestones. The structure and mechanism of DNA, combined with the knowledge of the human genome, paved the way for groundbreaking advancements, from gene editing via CRISPR, to gene therapies that improve the function of diseased or impaired cells through the delivery of genetic material.
As science started to uncover the potential of gene therapies, the importance of targeted delivery and efficient transgene expression became evident. This inspired researchers to seek various gene delivery strategies.
Delivering genetic material
Despite their pathogenic nature, viruses are the ideal vehicle for delivering genetic material. By introducing their genetic material into their hosts, viruses prompt host cells to express viral proteins or integrate viral DNA directly into the host genome. This discovery encouraged scientists to harness this mechanism by replacing viral genetic material with therapeutic DNA and RNA, giving rise to viral vectors.
Think of viral vectors as delivering the genetic material to tell the target cells what to do and when to do it. When the gene-based therapeutic is delivered to the target cell, it instructs the cell to produce antigens to evoke an immunogenic response from the body in the case of cancer or viral diseases.
Long-term therapeutic effects
Viral vectors deliver therapeutic DNA into the target cells with the goal of restoring gene expression that underlies genetic disorders. By studying the persistence of biodistribution, scientists have learned that virus-delivered genomes are detectable long after administration. This means the therapeutic effects are long-term, which reduces the need for subsequent dosing. Compared to non-viral gene therapies, such as the injection of naked DNA, viral vectors provide several advantages, including high transduction efficiency, higher rates of cellular entry, and substantial target protein expression to evoke the desired cellular machinery.
The last few years have seen eight viral vector therapies approved by FDA, including the first adenoviral vector-based gene therapy for the high-risk Bacillus Calmette-Guérin (BCG), a unresponsive non-muscle invasive bladder cancer, and adeno-associated virus being approved for spinal muscle atrophy (SMA), and more could be on the way3.
Given the growing influence of viral vector therapies in the field, evaluating the quality of viral vectors is crucial to ensuring efficacy and safety. Researchers and manufacturers focus on two criteria: vector copy number (VCN) and transgene expression. VCN indicates the average number of viral copies delivered to the target population, while transgene expression determines the efficiency with which the viral vector promotes gene delivery and its therapeutic effect. VCN and transgene expression can be inferred from the quantification of viral DNA and RNA in the target cell population, respectively. Therefore, efficient extraction of DNA and RNA is the key to establishing the quality attributes of the viral vector.
Preserving the genetic yield
Simultaneous extraction of DNA and RNA from the same biological specimen is helpful, especially when it comes to precious samples. Existing protocols achieve extraction by splitting the lysate into two portions, where DNAse is introduced to one half and RNAse A to the other. The main drawback of this method is that half of the DNA or RNA is lost in the process. SPRI technology, which utilizes paramagnetic beads to selectively bind nucleic acids of different sizes, is able to overcome this hurdle.
The SPRI workflow involves the separation of DNA from RNA followed by the subsequent purification of both in separate tubes. The critical step, in the beginning, is the adjustment of the crowding agent concentration to grant the beads preferential binding affinity to the larger DNA fragments, while the RNA remains in the supernatant. Once the DNA is transferred to a separate tube, the beads can be washed with ethanol to remove impurities, while the same is applied to the RNA transferred to another tube.
Automation for high throughput demand
While the manual extraction method described above is practical for small batches of samples, high-throughput processing calls for automation. A recent example was demonstrated by BridgeBio’s animal biodistribution studies, where researchers administered adeno-associated viral vectors (AAV) to different tissue types in mouse and non-human primate (NHP) models. DNA/RNA characterization was crucial to the researchers in determining the amount of gene therapeutic delivered to these tissues and the level of transgene expression.
Although column-based methods were previously used for extraction, SPRI offered many advantages in yield, turnaround time, and flexibility. “While both column-based and bead-based methods are reliable and reproducible, bead-based (SPRI) methods are more scalable for high-throughput analysis. In addition, the SPRI protocol gave more flexibility in terms of the tissue types we can lyse, whereas we needed different lysing kits for tissue samples from skeletal muscle and spinal cord,” Jeremy Rouse, a scientist at BridgeBio, explained.
Using SPRI technology also offered a safer alternative.
“Nucleic acid purification was one of the first skills I learned in the lab almost 25 years ago,” Rouse said. “Back then I used phenol and chloroform to extract nucleic acid from plants, which can have its own risks, Recently I worked on projects using SPRI technology to automate library preparation for NGS. That experience led me to using Beckman Coulter Life Sciences for our nucleic acid purification of animal tissue at BridgeBio.”
Confidence in analysis
Most importantly, the automated simultaneous extraction of DNA and RNA via SPRI technology made BridgeBio more self-reliant and confident in their viral vector analysis data. “Our team is now able to rapidly go from testing our gene therapy in animal models to evaluating its effectiveness. We don’t need to wait for a CRO to process our samples, which could take months. We can keep our team lean so that we can spend more time doing other research,” Rouse said.
This collaboration is only one example. DNA/RNA extraction can broadly shape the future of viral vector-based gene therapies by accelerating the quality assessment process. More importantly, the complete extraction of genetic material will improve the accuracy of impurity analysis by uncovering the precise amount of process-related impurities in the viral vector product. Elimination of these impurities, enabled by SPRI, can help overcome hurdles regarding the efficacy and safety of viral vector-based gene therapies.
As we reach the seventieth anniversary of the discovery of the double helix DNA structure, improvements in delivery systems such as viral vectors will be integral to successful targeting strategies in viral vector gene therapies, especially for rare genetic diseases with limited treatment options due to inadequate targeting.
“In my current role, I see firsthand where the understanding of rare genetic diseases can lead to improvements in many peoples’ lives, who 70 years ago there wouldn’t have been a diagnosis,” Rouse concluded.
- Watson, James D., and Francis HC Crick. “Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid.” Nature 171.4356 (1953): 737-738.
- International Human Genome Sequencing Consortium. “Finishing the euchromatic sequence of the human genome.” Nature 431.7011 (2004): 931-945.
Han Wei, Ph.D. is a Market Development Scientist at Beckman Coulter Life Sciences with a focus on building collaborative relationships with external partners. Prior to joining Beckman Coulter Life Sciences, she worked as a Research Associate and Postdoctoral Fellow at Indiana University School of Medicine. She received her Doctor of Philosophy in Biochemistry & Molecular Biology at the Graduate University of Chinese Academy of Sciences, Beijing, China, and a Master of Medicine in Toxicology at The Academy of Military Medical Sciences, Beijing, China. She also received a Bachelor of Medicine in Clinical Medicine (B.M.E.D., – equivalent to M.D.) at Southern Medical University in Guangdong, China.
Filed Under: Cell & gene therapy
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