A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells.

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

Mouse APOBEC3 expression in NIH 3T3 cells mediates hypermutation of AKV murine leukemia virus.

Mouse APOBEC3 (mA3) is a cytidine deaminase that can act on the single-stranded DNA reverse transcripts of retroviruses resulting in G→A hypermutation of proviral DNA. Many mA3 studies have used NIH 3T3 cells assuming that endogenous mA3 production was negligible. We developed a monoclonal antibody specific for mA3 that reveals detectable mA3 in NIH 3T3 cells and we demonstrate that AKV released from the cells undergoes G→A hypermutation. Inactivation of the mA3 gene abolished the deamination confirming that AKV hypermutation was mediated by mA3. The G→A mutations in AKV viral transcripts deviated from a normal distribution with all the mutations contained within only 20% of the transcripts. Single cell analyses revealed that the expression of mA3 in NIH 3T3 cells was limited to 20% of the cells, which likely accounted for the abnormal distribution of mutations. Endogenous NIH 3T3 mA3 was found to restrict AKV replication.

Latent murine leukemia virus infection characterized by the release of non-infectious virions.

Clonal cell lines derived from cultures infected with a polytropic MuLV release vastly different levels of infectious virions ranging from undetectable to very high. Low producing clones release an overwhelming proportion of non-infectious virions containing retroviral RNA but deficient in the Env protein. Non-infectious virion production is not due to an inability of the cells to support infectious MuLV production or to an inherent replicative defectiveness of the proviruses. Reinfection of the lowest producing lines with the polytropic or an ecotropic MuLV results in enormous increases in the specific infectivity of the released virions. This indicates a reversible state of retroviral latency characterized by the release of non-infectious virions that is likely the result of insufficient levels of Env protein required for infectivity. The latency state described here may have important roles in in vivo retroviral infections including alterations of the immune response and the production of defective interfering particles.

Endogenous retroviruses mobilized during friend murine leukemia virus infection.

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses.

Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety.

A total of 31 of thiourea derivatives was prepared reacting 3-(trifluoromethyl)aniline and commercial aliphatic and aromatic isothiocyanates. The yields varied from 35% to 82%. All compounds were evaluated in vitro for antimicrobial activity. Derivatives 3, 5, 6, 9, 15, 24 and 27 showed the highest inhibition against Gram-positive cocci (standard and hospital strains). The observed MIC values were in the range of 0.25-16 µg/ml. Inhibitory activity of thioureas 5 and 15 against topoisomerase IV isolated from Staphylococcus aureus was studied. Products 5 and 15 effectively inhibited the formation of biofilms of methicillin-resistant and standard strains of Staphylococcus epidermidis. Moreover, all obtained thioureas were evaluated for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses. Compounds 5, 6, 8-12, 15 resulted cytotoxic against MT-4 cells (CC50 ≤ 10 µM).

Antimicrobial and anti-biofilm activity of thiourea derivatives incorporating a 2-aminothiazole scaffold.

A series of new thiourea derivatives of 1,3-thiazole have been synthesized. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. Compounds were also tested for their in vitro tuberculostatic activity against the Mycobacterium tuberculosis H37Rv strain, as well as two 'wild' strains isolated from tuberculosis patients. Compounds 3 and 9 showed significant inhibition against Gram-positive cocci (standard strains and hospital strain). The range of MIC values is 2-32 µg/mL. Products 3 and 9 effectively inhibited the biofilm formation of both methicillin-resistant and standard strains of S. epidermidis. The halogen atom, especially at the 3rd position of the phenyl group, is significantly important for this antimicrobial activity. Moreover, all obtained compounds resulted in cytotoxicity and antiviral activity on a large set of DNA and RNA viruses, including Human Immunodeficiency Virus type 1 (HIV-1) and other several important human pathogens. Compound 4 showed activity against HIV-1 and Coxsackievirus type B5. Seven compounds resulted in cytotoxicity against MT-4 cells (CC50<10 µM).

Incorporation of mouse APOBEC3 into murine leukemia virus virions decreases the activity and fidelity of reverse transcriptase.

APOBEC3 proteins are restriction factors that induce G→A hypermutation in retroviruses during replication as a result of cytidine deamination of minus-strand DNA transcripts. However, the mechanism of APOBEC inhibition of murine leukemia viruses (MuLVs) does not appear to be G→A hypermutation and is unclear. In this report, the incorporation of mA3 in virions resulted in a loss in virion reverse transcriptase (RT) activity and RT fidelity that correlated with the loss of virion-specific infectivity.

Analysis of two monoclonal antibodies reactive with envelope proteins of murine retroviruses: one pan specific antibody and one specific for Moloney leukemia virus.

Many monoclonal antibodies (MAbs) reactive with various proteins of murine leukemia viruses (MuLVs) have been developed. In this report two additional MAbs with differing and unusual specificities are described. MAb 573 is reactive with the envelope protein of all MuLVs tested including viruses in the ecotropic, xenotropic, polytropic and amphotropic classes. Notably, MAb 573 is one of only two reported MAbs that react with the envelope protein of amphotropic MuLVs. This MAb appears to recognize a conformational epitope within the envelope protein, as it reacts strongly with live virus and live infected cells, but does not react with formalin-fixed or alcohol-fixed infected cells or denatured viral envelope protein in immunoblots. In contrast, Mab 538 reacts only with an epitope unique to the envelope protein of the Moloney (Mo-) strain of MuLV, a prototypic ecotropic MuLV that is the basis for many retroviral tools used in molecular biology. MAb 538 can react with live cells and viruses, or detergent denatured or fixed envelope protein. The derivation of these antibodies as well as their characterization with regard to their isotype, range of reactivity with different MuLVs and utility in different immunological procedures are described in this study.