Viral vectors for gene therapy
Although the first successful treatments are just know being demonstrated in clinical trials, gene therapy will have a huge impact on many life-threatening diseases. Viral vectors are the most efficient gene delivery vehicles; however, their manufacturing is complex, requiring the use of animal cell cultures. Currently, they are the basis for numerous gene therapy clinical trials.
The Animal Cell Technology (ACT) Unit has a track record in the development of several viral vectors for gene therapy namely, retrovirus and lentivirus, adenovirus, adeno-associated virus, and more recently baculovirus. Each of these viral vectors each present several advantages and disadvantages from the safety, efficacy, and manufacturing perspective, which are being addressed by our group to further improve them.
Retrovirus and lentivirus
Retroviral vectors, based on Murine leukaemia virus, were the first viral vectors to become available and used in clinical trials. One of the characteristics of retroviral vectors is their ability to integrate the genetic material transferred into the host-cell resulting in long-term transgene expression. More recently another class of retrovirus was developed from HIV into gene therapy vectors, these were denominated lentiviral vectors. They present the additional advantage of being able to transduce quiescent cells. Both vectors present however several difficulties in their manufacturing, as they are very labile, and the cell productivities are low.
Current research at the ACT Unit focuses on the development of enhanced cell lines and analysis of the metabolic requirements for vector production. Additionally, we develop production and purification processes for both vectors.
Adenovirus
Adenoviral vectors are presently the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, infection diseases or neurological disorders. Human adenoviruses (hAdV) are generally the chosen prototype vector backbone.
A platform for production, purification, and storage was developed by the ACT Unit, where the constraints on viral production and the so called ‘cell density effect’ were thoroughly analyzed and elucidated in HEK 293 cells. Presently, alternative human cell lines are being studied.
One of the major hurdles regarding the use of hAdV is the pre-existent immunity memory that may limit the efficiency of transgene expression in humans. To circumvent this, non-human adenovirus vectors such as Canine Adenovirus Vectors type 2 are also being developed and studied by our group. The restrict experience in the field with these vectors requires the establishment of an entire process development suitable for GMP manufacturing. Current objectives include the development of canine transcomplementing cell lines that support the amplification of the vectors (E1 deleted and helper dependent vectors) and the development of upstream and downstream processes.
Adeno-associated virus
Along with their strong clinical safety profile, adeno-associated viruses (AAVs) can transduce both dividing and non-dividing cells and achieve long term transgene expression. Nonetheless, AAVs show a wide tissue tropism, which constitutes the major limitation for their use for some applications, especially for systemic application.
The ACT Unit is investing on new approaches towards targeting AAVs to cells of therapeutic interest, with the final goal of using them as improved vehicles for gene therapy.
Baculovirus
For several decades, the baculovirus-insect cell system has been used for industrial and research purposes, maturing into a versatile technology for producing biopesticides, recombinant proteins and vaccines. Given their safety and large genomic capacity, the baculovirus (BV) itself is beginning to be addressed as a gene therapy vector. However, from a bioprocessing viewpoint, a critical bottleneck is the decreased specific productivities obtainable from high cell density culture, with implications for overall process costs.
Current research at the ACT Unit on this subject includes a combination of experimental and computational tools to assess the metabolic determinants of BV production. Furthermore, we have been developing processes for BV purification. The distinct properties of this virus, in particular surface heterogeneity and non-spherical rod-shaped structure, represent major challenges in the development of robust downstream strategies to produce clinical grade preparations.
