Archive 21/1/27

In gene therapy using Adeno-associated virus (AAV), it has been suggested that there are some differences in AAV capsids in terms of tissue targeting ability, and it has been told that glycans may be involved in he AAV’s tissue targeting ability. Such arguments have been around for quite some time.
For example, the following papers are references.
https://www.nature.com/articles/gt201316
https://www.jbc.org/article/S0021-9258(20)51734-1/fulltext
https://www.jbc.org/article/S0021-9258(20)48853-2/fulltext
https://jvi.asm.org/content/80/18/9093

Specifically, it has been reported that AAV-1 and AAV-6 bind to Sialic acid, AAV-2 binds to Heparan sulfate, and AAV-9 binds to Galatose.
But, is it true?
To this blog admin., it is a kind of unbelievable thing that capsid proteins have lectin-like functions?

Toxicology tests using hepatocytes have come to be widely used as an alternative way for testing drug toxicology using animals. The problem with using hepatocytes in toxicology tests is that it is difficult to supply large quantities of hepatocytes with the same characteristics. Therefore, a group from the National Institute of Child Health and Development proposes a method to create hepatocytes from pluripotent stem cells (iPSC, ESC) and enrich highly uniform hepatocellular populations from heterogeneous cell populations using ammonia.
https://www.biorxiv.org/content/10.1101/2020.03.20.999680v1.full

After ESC is differentiated into hepatocytes, an ammonia treatment is performed for 2 days to select uniform hepatocytes that are resistant to ammonia toxicity. 70-80% will die at this stage. Surviving hepatocytes are cultured and proliferated on MEF-feeder. Hepatocytes selected in this way multiply about 30 times in 190 days. The detailed mechanism of ammonia toxicity is not known, but it is thought that ammonia ions compete with potassium ions and eventually die with intracellular and/or extracellular pH change. ALB, AFP, CYP3A4, CPS1, and OTC genes are highly expressed in ammonia-selected hepatocytes, and ALB and AFP decrease with the passage. CPS1 and OTC are genes involved in the metabolism of ammonia. CYP3A4 is one of the enzymes that metabolizes unwanted biological molecules.
The same is possible with hepatocytes made from iPSC.

The differentiated cells from iPSC and /or ECS were further cultivated and propagated in the ESTEM-HE medium (GlycoTechnica, Ltd.) .

Hepatocytes are essential for toxicology testing of pharmaceuticals in vitro. Hepatocyte strains such as HepG2, Huh7, THLE-2, PLC-PRF-5, and AML-12 are used. A group from the National Center for Child Health and Development has established a new immortalized hepatocytes  named HepaMN from from a liver associated with biliary atresia. Immortalization was performed by inoculation with CDK4, cyclin D1, and TERT.
https://www.nature.com/articles/s41598-020-73992-3

Characteristics of HepaMN strains:
Established from untransformed hepatocytes, and has normal liver function and diploid chromosomes,
Expression of albumin gene at the same level as HepaRG,
Showing the cell morphology of hepatocytes,
Induces stable cytochrome P450 3A4 (CYP3A4) and shows normal metabolic effects,
Efficiently proliferates and is stable even in very long passages,
As a result, succeeded in providing a new and useful hepatocyte strain for toxicology testing.

HepaMN can be effectively cultured by EMUKK-15

Existing Pfizer and Modelna vaccines for the new coronavirus (SARS-CoV-2) are mRNA type, but the following group is evaluating prototype vaccines using adeno-associated viral (AAV).
https://www.biorxiv.org/content/10.1101/2021.01.05.422952v3.full.pdf

Therapeutic gene transfer using AAV is recognized as a highly safe method, and Glybera, Luxturna, Zortensma etc. have already been approved by the FDA and EMA as gene therapeutics.
Vaccines using two types of AAV have been prototyped. AAVCOVID-19-1 (AC1) encodes a full-length S protein, which expresses an antigen S protein on the cell membrane of an infected cell. Another, AAVCOVID-19-3 (AC3), encodes part of the S protein, and the antigen takes the form of a secretory protein.

The evaluation was carried out with mice and nonhuman primates, and the immune response was retained for about 5 months with a single doze. There is no problem with storage at room temperature for one month, and vaccines are amenable to large scale production because the methodology has been established. As an effect, AC1 seems to be higher than AC3.

However, the blog Admin has some concerns that the antigen genes are incorporated into cells.

It has been pointed out that mutations in the new coronavirus (SARS-CoV-2) are behind infection explosions in the UK and South Africa. A number of mutations have occurred, but a mutation common to these has already been shown to be N501Y.
A group of The University of Texas Medical Branch, Galveston TX has reported on how the mutation affects the effects of Pfizer’s vaccine, Tozinameran (BNT162b2, a nucleoside modified RNA vaccine).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805448/

A comparative experiment using N501 and Y501, which is the genetic background of N501,  was done by measuring  50% plaque reduction neutralization titers for N501 and Y501 viruses using sera from 20 people collected 2 weeks and 4 weeks after administration of the vaccine. As a result, it was reported that almost the same neutralization titers were obtained.