Howe, Geoffrey; (2025) Engineering Biology Approaches to Improving Viral Vector Manufacturing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Viral vectors are synonymous with potentially curative therapeutic gene transfer. But these are expensive to manufacture which restricts equitable global patient access. Human embryonic kidney cells (HEK) are widely used as host cells for producing viral vectors and vaccines, despite not being naturally evolved for this purpose. Similarly, viral vectors themselves have not evolved for ease of manufacturability as gene delivery vehicles. To address host cell limitations, HEK cells were engineered with a staphylococcal nuclease (NuPro cells) and grown in suspension serum-free cell culture as a highly scalable system for autonomic, cell-mediated DNA impurity reduction. To address viral limitations, synthetic biology was employed to redesign the AAV5 MAAP protein to enhance AAV5 secretion as a foundational step toward lysis free bioprocessing of this poorly secreted serotype. These engineering biology solutions establish a novel framework for improving viral vector manufacturing at scale. Lentivirus is a proven cell and gene therapy tool which requires costly process additions to remove DNA impurities from process streams. Lentivirus production using suspension NuPro host cells preserved lentivirus yield performance whilst effecting an 89% reduction in total DNA impurities. Adenovirus is a proven vaccine and gene therapy platform but due to its lytic harvest step biomanufacturing is compromised by the need to remove DNA impurities from process streams. Traditional and low-MOI methods were trialled in suspension NuPro cells and in unmodified suspension cells. While secreted nuclease activity was observed, DNA clearance after Adenovirus production did not manifest. However, Low-MOI approaches in suspension unmodified cells reduced DNA impurities while reducing the amount of starting virus material required 800-fold. Adeno-associated virus (AAV) is surging in popularity as a gene therapy tool and its scaled manufacturing is similarly burdened by the presence of DNA impurities. Production of AAV5 and AAV9 using the suspension NuPro host cells marginally improved yield performance whilst also effecting a 60% reduction in high molecular weight DNA impurities. AAV is generally harvested via cell lysis due to generally insufficient secretion across serotypes. The ability to boost secretion of a given serotype is therefore potentially beneficial to AVV bioprocessing. The fundamental biology of the AAV-encoded MAAP protein was both investigated and harnessed as an engineering tool to re-program AAV5 secretion. Rational MAAP engineering successfully achieved an 8.19-fold increase in AAV5 secretion. This is the first time MAAP has been rationally re designed to help establish an engineering toolbox to control AAV secretion.

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