Axial — Observations #39. Get these analyses to your inbox —…
Tags: Venture Capital, Biotechnology, Investing, Healthcare, Medicine

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Observations #39

A set of ideas and observations from a week’s worth of work analyzing businesses and technologies.

AAV manufacturing

Biomanufacturing of adeno-associated virus (AAV) is composed of 5 main steps:

Cell culture (insect cells, HEK293) — HEK293 is the standard Culture (suspension, 3D adherent, 2D) — suspension Vector production (transient expression, stable cell line) — transient and takes 2–3 days Recovery (filtration, centrifugation, TFF) — filtration and TFF Purification (ultracentrifugation, chromatography) — chromatography

The first AAV gene therapy approved was Glybera in 2012 for LPLD in the EU followed by FDA approval of Luxturna in 2017 for IRD and Zolgensma in 2019 for SMA. As more of these medicines come through the clinic, more manufacturing capacity is required to expand to indications with larger patient populations.

Currently, two methods are the standard for AAV manufacturing — HEK293 and Sf9:

HEK293 cells that are adapted for suspension and are transfected with plasmid DNA. A master cell line is required along with plasmid DNA and transfection agents that increase COGS for each batch. Stoichiometry and expression become important when there are multiple plasmids used (helper and transfer).

Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system (BEVS) for transfection. During a manufacturing run, baculoviruses (one for rep/cap and the other for the AAV genome) with the transfer genes to generate the AAV vectors are infused with the Sf9 cell culture. Each baculovirus requires a master line, which increases the upfront investment required versus HEK292. However, yields in an Sf9 system are significantly higher.

A major theme in gene therapies is that these processes are the actual product. Ultimately, the goal is to develop cell-lines that maximize viral titers (i.e. produce fewer empty capsids) and minimize upfront development costs (a batch can cost upwards of $500K). However, the AAV manufacturing field has 3 major problems to surmount:

Developing stable cell lines to increase yields — the current standard for vector production relies on transient transfection, mainly to reduce costs Minimize the variability between batches, serotypes, and cargos — depending on the serotype and what is being delivered can lead to wide variations in outcomes across manufacturing runs Reduce the number of empty capsids produced — cell lines produce many non-functional empty capsids that need to be separated from functional capsids Improve purification — recovering AAV vectors from culture supernatant versus cell lysate. Moreover, generic processes particularly around purification are needed to apply across multiple vectors.

Over time, there is a large opportunity to design and scale vector-specific manufacturing processes. With more than 12 AAV serotypes, 100s of natural variants, and many more synthetic versions, the same focus and specificity required for drug development is needed in biomanufacturing.

Source: Piper Jaffray

iPSC-derived cell therapies

Induced pluripotent stem cells (iPSC) have the ability to differentiate into any cell type and have the power to self-renew. These features make them particularly compelling for regenerative medicine and allogeneic cell therapies.

The key work was led by Shinya Yamanaka at Kyoto University to discover and use 4 reprogramming factors (OCT4, SOX2, MYC, and KLF4) to induce pluripotency from mouse embryonic or adult fibroblasts — https://www.cell.com/cell/fulltext/S0092-8674(06)00976-7 Before this work was published in 2006, the stem cell field relied on human embryonic stem cells (ESC), which are difficult to source and historically has controversy surrounding the research. Once Yamanaka published this research, it became clear that iPSCs can solve these sourcing and ethics problems that have slowed down progress in the field. Rather than sourcing an ESC for each barch, one can create a iPSC master cell line.

Before 2006, some early success of ESCs in clinical trials — (1) https://pubmed.ncbi.nlm.nih.gov/29553577/ and (2) https://pubmed.ncbi.nlm.nih.gov/27144379/ have set up iPSC-derived cell therapies to have some sort of precedent and hopefully get translated into new medicines sooner rather than later — https://ir.fatetherapeutics.com/news-releases/news-release-details/fate-therapeutics-announces-fda-clearance-ind-application-ft596

Like most new inventions, iPSCs face a host of new challenges to solve. Currently, iPSCs have a longer road to travel and a few key problems to solve:

Teratoma formation — pluripotent stem cells can form teratomas in a patient due to the cancer risk of the 4 Yamanaka factors. A useful strategy is to purge residual iPSCs before a transplant. Carcinogenicity — related to (1), expression of the 4 factors, in particular MYC and KLF4, increase the odds of tumor formation. Finding substitute genes that reduce this risk but still induce pluripotency is needed: Nanog and LIN28 might be able to substitute for MYC and KLF4. Genomic instability — there is evidence that reprogramming a cell itself can lead to generic instability (i.e. amplifications, deletions) as well as chromosome and epigenetic abnormalities. This is still an important area of research with a focus on the role of p53 in reprogramming. Immunogenicity — iPSCs can lead to immune rejection depending on the tissue and minor antigen presented. One strategy is to use genome engineering to knockout diverse HLA genes and activate immunomodulatory genes to develop hypoimmunogenic iPSCs (i.e. the basis of Sana) — https://www.pnas.org/content/116/21/10441.abstract Quality control — manufacturing iPSCs consistently and reproducibly is needed. Better tools to isolate, select, and expand certain iPSC lines are needed. Increase the efficiency of differentiation — new factors and differentiation protocols are needed to reduce the number of residual iPSCs within a batch and hopefully reduce the time from months to weeks

Ultimately, iPSC-derived cell therapies have the potential to create master cell lines that can be used off-the-shelf to treat a large number of patients from oncology to regeneration.

Source: Fate Therapeutics

AlphaFold

The recent DeepMind AlphaFold result to predict protein structures in CASP14 is a massive breakthrough and validation for the premise of using “cheap” biological data to train models — https://predictioncenter.org/casp14/zscores_final.cgi This graph does a great job to anchor how much progress has been made:

What is Life? cont.

In Chapter 5 (Delbruck’s Model Discussed and Tested) of What is Life?, Schrödinger discusses the features that increases the stability of a molecule with the goal to understand how genetic information is stored: