Moving Towards Genome Integrity Evaluation of Gene Therapy Viral Vectors

David Dobnik, PhD- Senior Research Associate at National Institute of Biology, Chief Scientific Officer at Niba Labs Ltd.

Gene therapy is a rapidly developing field that holds promise for the treatment of a variety of genetic disorders. One of the most important components of gene therapy is the use of viral vectors to deliver therapeutic genes into target cells. These vectors are usually derived from naturally occurring viruses that have been genetically engineered to carry the therapeutic gene.

The use of viral vectors in gene therapy raises significant safety concerns, particularly with regard to drug safety. Viral vectors can cause severe immune reactions, especially because they are administered at relatively high concentrations. The first human death in the field of gene therapy dates back to 1999,¹ which was a setback for the development of such drugs. Scientists have made great efforts to find new viral vectors with the lowest possible immunogenicity. One such virus is adeno-associated virus (AAV), which does not cause symptoms in the human body.² Recombinant AAVs are currently one of the most widely used vectors for gene therapy. Although their use seems to be relatively safe, they can still cause serious side effects, including hepatotoxicity and neurotoxicity.³ Recently, deaths have been reported after the administration of a drug approved for the treatment of spinal muscular atrophy.⁴ Since the amount of viruses administered into the body during gene therapy treatment is relatively high (about 1e+14 vector genomes/kg), one of the solutions for a lower immune response would be to reduce the number of vectors that need to be injected into the patient.

Currently, even the final drug product of the highest purity may consist of different populations of capsids, including full capsids, empty viral capsids, and capsids that are not properly filled, i.e., they may contain only fragments of the intended recombinant vector genome or fragments of other nucleic acid impurities. During characterization, much emphasis is placed on evaluating the ratio of full to empty viral particles to optimize the process and maximize the number of full particles in the final drug product. Evaluating the ratio of full to empty viral vectors for gene therapy is an important step to ensure its safety and efficacy, as the immune response is related to the presence of capsids (empty or full) and their content.^5,6 The ratio of full to empty vectors refers to the proportion of viral vectors that carry the therapeutic gene (full) compared to those that do not (empty). It is important to have a high proportion of full vectors, as empty vectors may not provide therapeutic benefits and could also pose potential safety risks, as mentioned above. There are several ways to evaluate the ratio of full to empty vectors for gene therapy (Table 1).

Analytical ultracentrifugation (AUC) is currently still most widely accepted as the gold standard for evaluating the full-empty ratio.⁷ AUC is a powerful analytical tool that allows the separation and characterization of macromolecules based on their size, shape, and interactions. It can provide information on the size, shape, and interactions of viral vectors that can be used to evaluate their full-empty ratio. AUC uses a high-speed centrifuge to separate particles based on their sedimentation coefficient, which is a measure of particle size, density, and shape. Particles that are larger or have a different shape or density sediment at different rates, allowing for the separation of different particle populations. By measuring the sedimentation of viral vectors, AUC can determine the proportion of full vectors in the population. AUC is a highly sensitive technique that can detect small changes in the size and shape of viral vectors. It can also be used to detect the presence of empty vectors, which are smaller and have a different shape and density than the full vectors.⁸ It should be noted that AUC is a complex and expensive procedure that requires specialized equipment and trained personnel to use. It is not always the most accessible or convenient method for evaluating the ratio of full to empty viral vectors, but it is a useful tool for the research and development of gene therapy products. AUC is well suited for purified samples but not so much for testing the in-process samples. New variants are also currently being developed (e.g., gradient density AUC),⁹ which allow for more detailed analysis and better separation, although low throughput and complex analysis remain a major challenge.

Time is of great importance when analyzing in-process samples, thus new developments focus on ease of use and time-efficient analysis. One such approach is size exclusion chromatography with multi-angle light scattering (SEC-MALS),¹⁰ however, its challenge is that it is not able to distinguish partially filled particles, thus those are merged with the population of full particles (e.g., two half-length genomes each in individual particle would appear as one full and one empty particle after the analysis).

The ability to separate different capsid populations and determine the presence of partially filled capsids presents another challenge associated with viral vectors, namely the presence of partially filled particles. None of the methods used to determine the ratio of full to empty particles can determine and define the actual content of these capsids. Although the expected content of full particles is the full-length genome, it is becoming clear that this is not always the case. Primarily because the resolution of the methods used to assess the full-empty ratio does not extend to the base pair (or even 100 bp) level, it is expected that a variety of different lengths of vector genome are present in the capsids. As Rahul Sheth reported in a talk at the recent ARM’s AAV Analytical Characterization Workshop, after affinity purification and separation of full and empty particles, there is a population of empty particles that coelutes with a fraction of the full particles. This would explain why the final drug product still contains some of the empty particles if it was not properly purified. Ultimately, the empty particles may not even be completely empty, but due to the poor resolution of analytical methods, it is difficult to evaluate this fact.

This brings us to one of the emerging attributes of viral vectors, namely the integrity of the viral genome. The recombinant viral genome contains all the information required for the therapeutic effect of the manufactured construct (e.g., promoter, enhancer, gene of interest, polyA tail). If the viral genome is not complete, no therapeutic effect can be expected; moreover, such fragments may generate neoantigens in cells, leading to unexpected immune responses.

There are only a limited number of analytical methods for evaluating the integrity of the vector genome that focus on identifying the actual sequence rather than just the presence of fragments and that can provide good insight into the population of vector genome fragments encapsidated in viral particles. The first method is long-read sequencing. Long-read sequencing is a technique that allows sequencing of much longer DNA segments than traditional short-read sequencing methods. Two popular systems for long-read sequencing are PacBio and MinION technology. This approach determines the exact sequence of the viral genome fragment population and can also detect mutations, deletions, or other changes in the viral genome or reveal nucleic acid impurities. The PacBio system uses a technology called single-molecule real-time (SMRT) sequencing, which allows sequencing of individual DNA molecules. This system can generate read segments up to several kilobases in length and is characterized by high accuracy. This system has already been shown to be highly informative in AAV vector sequencing.^11,12 The MinION system uses a technology called nanopore sequencing, in which DNA molecules are passed through a nanopore and the changes in electrical current are measured as the DNA passes through the pore. This system can produce reads up to tens of kilobases long in a short time, and the technology is easy to use. The MinION system is particularly useful for real-time sequencing and rapid identification of pathogens/contaminants and can also serve as a tool for characterizing vector genomes. Both the PacBio and MinION systems are powerful tools for long read sequencing, each with their own strengths and weaknesses. A common problem with both systems is bias toward shorter reads, but this can be overcome to some extent by implementing appropriate controls that allow normalization of results to correct for sequencing bias.¹¹

Multiplex digital PCR (dPCR) is another emerging technique that has great potential for widespread application in genome integrity assessment.¹³ The distinguishing feature of dPCR is its ability to provide absolute quantification without standard curve. Currently, various dPCR platforms are used in the field of gene therapy to quantify the vector genome titer and specific nucleic acid impurities (e.g., host cell DNA, plasmid DNA). For this purpose, PCR amplification targets a relatively small region of the sequence, so the vector genome titer result is not indicative of the amount of full-length vector genome, but rather of the presence of target sequence. Drug efficacy and potency depend on a fully functional vector genome, so the vector genome titer is not very informative in this regard. Fragments of the vector genome cannot provide a functional product, so it is important to minimize their presence. The only truly quantitative option to determine the amount of full-length vector genomes at the moment is multiplex dPCR. This idea was first published in 2019¹³ by implementing a duplex reaction with targets in two distant regions of the vector genome. With the development of new dPCR platforms that allow higher levels of multiplexing, the potential of such analyses became even better. Multiplex results using more targets per genome provide an additional layer of information that can be used to optimize the production process and improve the final drug product in terms of full-length genome content.¹⁴

The principle of dPCR is to partition the sample into thousands of low-volume reactions. By implementing multiple occupancy analysis of each partition, we can calculate the titer for each of the fragments. In this way, we obtain a quantitative result for each fragment from a population of possible fragments, including full-length vector genomes (Figure 1). In conjunction with Figure 1, the results of the simplex analysis (vector titers) are shown in Table 2. As can be seen from the example in Figure 1, the concentration of the full-length vector is approximately four times lower than that obtained by the simplex assay for four different individual regions (Table 2). The additional information provided by such a multiplex approach is the concentration of the different fragment populations. In the example (Figure 1), the three most abundant particle populations are the full-length vector and the first and second halves of the vector genome. These results suggest that the packaging of the vector genome in the capsid may be influenced by the sequence in the middle region of the construct. Such information may provide valuable clues for optimizing the sequence or the upstream process, which in turn would lead to a better yield of full-length vectors. One of the main advantages of such a multiplex dPCR approach is to evaluate the integrity of the vector genome with a high-throughput method. Taking this information into account, the production process can be improved such that the final drug product contains more full-length genomes, which would likely increase the efficacy of the drug product. Thus, the final dose of capsids received by the patient would need to be lower, and the drug would be safer for the patient because fewer capsids would be administered into the body, reducing the possibility of a severe immune response. In addition to a better-characterized product, manufacturers could also lower the cost of production per dose.

The use of viral vectors in gene therapy holds great promise for treating a broad range of genetic disorders. Rigorous and meaningful quality controls are needed to ensure the safety and efficacy of gene therapy viral vectors. Our suggested approach would be to assess the integrity of the viral genome, and as described above, multiplex dPCR is currently the best option for such characterization, especially when focusing on more extensive testing of different process samples where the use of long-read sequencing would not be feasible or cost-effective.

Acknowledgment

The funding for the preparation of the manuscript were received by Slovenian Research Agency (contract numbers: P4-0407 and L4-3180). The results on genome integrity multiplex were kindly provided by Niba Labs.

References

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Author Biography

David Dobnik, PhD, has been developing new nucleic acid detection/ quantification approaches since 2007. From 2016, he has been leading the development and tech-transfer projects for pharmaceutical companies focused on the field of gene therapy, helping their process development efforts with characterizations of viral vectors. Lately, most of his work has been focused on new approaches for genome integrity evaluation.

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