Adeno-Associated Viruses (AAVs) are quickly becoming one of the most applied vectors for gene therapy applications. In the recent years three new AAV-based gene therapies have been approved by U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The regulatory bodies require accurate and reliable characterisation of the clinical grade viral vectors during and after production. Analytic methods measuring the purity, potency and safety of the product support the up-stream and down-stream processes during the production and are used for final-drug substance characterisation. Median Tissue Culture Infectious Dose (TCID50) is a well-established method for measuring the infectious titer of a virus. Here, an assay for determining the infectious titer of AAVs, which has previously been used to characterise the existing AAV2 Reference Standard Material (AAV2RSM) was set up and optimised for research use at Kuopio Center for Gene and Cell Therapy (KCT). The assay utilizes the HeRC32-cell line, a HeLa clone, that stably expresses AAV Rep and Cap -proteins and in presence of adenovirus, enables the replication of recombinant AAV-vectors. The cells were grown in 96-well plates and infected with 10-fold dilution series of AAV vectors (AAV2 and AAV6) using human adenovirus type 5 as the co-infector. 72 hours post infection the vector genome replication of AAV was detected with quantitative PCR (qPCR). Thresholds for qPCR determined copy number and cycle threshold (Ct) were set and used for the determination of infection-positive wells. The 50-percent endpoint was observed and used to calculate the infectious titer according to the Spearman-Kärber method. The assay was set up and optimised with the AAV2 Reference standard material (AAV2RSM) using two different primer-probe sets (targeted sequences were; SV40 polyadenylation signal and AAV inverted terminal repeats (ITRs)). Plates infected with AAV2RSM were analysed separately with both primers resulting in mean infectious titers of 8.07 ± 3.13 x 108 TCID50 Infectious Units (IU) / mL (n = 9) and 1.27 ± 0.464 x 109 TCID50 IU/mL (n = 9) for SV40 and ITR, respectively. After the assay was set up with the AAV2RSM, an in-house AAV6 product was analysed with the ITR primers yielding 6.09 ± 3.94 x 109 TCID50 (IU) / mL (n = 5). The assay protocol was successfully set up for research use at the KCT laboratory. Improvements were added to the original protocol to increase assay robustness, accuracy and precision as well as to minimize the possibility of over-estimation of the infectious titer. The assay can be further optimised for a particular therapeutic AAV product in the research laboratory or technology transferred to a production facility for optimisation and validation for the analytics needs of a production pipeline.

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 angstrom resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.

After the discovery and characterization of the adenovirus in the 1950s, this prevalent cause of the common cold and other usually mild diseases has been modified and utilized in biomedicine in several ways. To date, adenoviruses are the most frequently used vectors and therapeutic (e.g., oncolytic) agents with a number of beneficial features. They infect both dividing and nondividing cells, enable high-level, transient protein expression, and are easy to amplify to high concentrations. As an important and versatile research tool, it is of essence to understand the limits and advantages that genetic modification of adenovirus vectors may entail. Therefore, a retrospective analysis was performed of adenoviral gene therapy constructs produced in the same laboratory with similar methods. The aim was to assess the impact of various modifications on the physical and functional titer of the virus. It was found that genome size (designed within "the 105% golden rule") did not significantly affect the physical titer of the adenovirus preparations, regardless of the type of transgene (e.g., immunostimulatory vs. other), number of engineered changes, and size of the mutated virus genome. One statistically significant exception was noted, however. Chimeric adenoviruses (5/3) had a slightly lower physical titer compared to Ad5-based viruses, although a trend for the opposite was true for functional titers. Thus, 5/3 chimeric viruses may in fact be appealing from a safety versus efficacy viewpoint. Armed viruses had lower functional and physical titers than unarmed viruses, while five genomic modifications started to decrease functional titer. Importantly, even highly modified armed viruses generally had good titers compatible with clinical testing. In summary, this paper shows the plasticity of adenovirus for various vector, oncolytic, and armed oncolytic uses. These results inform future generations of adenovirus-based drugs for human use. This information is directly transferable to academic laboratories and the biomedical industry involved in vector design and production optimization.

Gene therapy provides an ideal potential treatment for intervertebral disk degeneration by delivering synthetic microRNAs (miRNAs) to regulate the gene expression levels. However, it is very challenging to deliver miRNAs directly, which leads to inactivation, low transfection efficiency, and short half‐life. Here, Agomir is loaded in hydrogel to construct a gene‐hydrogel microenvironment for regulating the synthesis/catabolism balance of the tissue extracellular matrix (ECM) to treat degenerative diseases. Agomir is a cholesterol‐, methylation‐, and phosphorothioate‐modified miRNA, which can mimic the function of miRNA to regulate the expression of the target gene. Agomir874 that mimics miRNA874 is synthesized to down regulate the expression of matrix metalloproteinases (MMPs) in nucleus pulposus (NP). At the same time, a polyethylene glycol (PEG) hydrogel is synthesized through Ag‐S coordination of 4‐arm PEG‐SH and silver ion solution, which has injectable, self‐healing, antimicrobial, degradable, and superabsorbent properties and matches perfectly with the mechanism of intervertebral disk. By delivering Agomir‐loaded PEG‐hydrogel to a degenerative intervertebral disk, a gene‐hydrogel microenvironment is constructed in situ, which reduces the expression of MMPs, regulates the synthesis/catabolism balance of ECM in the NP of the intervertebral disk, and improves the tissue microenvironment regeneration.

Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.

Aspartylglucosaminuria (AGU) is a rare lysosomal storage disorder causing developmental delay, intellectual disability, and eventual death. A distinct feature in AGU is iron accumulation within the thalamus. Our aim is to demonstrate that susceptibility-weighted images (SWI) could be used as an MRI biomarker to evaluate the response within the AGU population to newly evolving treatments. SWI from 16 patients with AGU and 16 age-matched controls were used in the analysis. Thalamic volume with an iron accumulation was identified using a permutation test. Group differences were investigated for both the complete thalamus and the iron accumulation regions. Group-wise age correlation within these volumes were assessed with analysis of variance and multivariate regression. We found a statistically significant and large difference (p-value = 0.01, Cohen's D = 0.97) for the whole thalamus comparison and an even greater difference in the iron accumulation regions (p-value <0.01, Cohen's D = 3.52). Furthermore, we found strong evidence for iron accumulation as a linear function of age with R-2 = 0.65 only for AGU. The statistical analysis of SWI provides tools for assessing the degree of iron accumulation. This method could be used to study the response to treatments, in that a successful treatment would be expected to result in a decline in iron accumulation.