1. Outline of Nucleic Acids Delivery

Background

It is well known that some biopharmaceutical companies have withdrawn from siRNA research probably because the liver continues to be the only target of siRNAs. Accordingly, every venture needs to compete in a small market related to the liver. GenAhead Bio would like to introduce a novel siRNA delivery platform, which expands the target organ outside the liver.　
In the Liver, it is well known that Liver-specific ligand works well to deliver siRNA, therefore it should be possible then to use the other receptor-ligand pair to deliver siRNAs especially outside the Liver. To study this possibility, we started to use antibodies in 2012.

 

Particle size might hinder CD71-targeting potential

GenAhead Bio had screened and selected anti-CD71 antibody as a targeting ligand for leukocytes and muscular tissues. GenAhead Bio also had the efforts in minimizing the platform. Apart from the liver and tumor, the pore size in normal vasculature are very small thereby leading to failure to reach the target organ across the endothelium even when CD71 expressions were very high. One of the most small platform is anti-CD71 Fab’ – siRNA conjugate in which we used HPRT siRNA as a reporter gene.

 

Affinity / Resistance against natural Tf

Ligand-receptor binding was maintained after the conjugation with an EC50 value of 29 nM. This affinity is typical as monovalent antibody. Bivalent antibody sometimes shows toxicities resulted from crosslinking of cell-surface receptors. Compatibility with endogenous ligands (different epitope) is helpful to avoid interference of receptor function, and also is advantageous to work in vivo in the presence of abundant transferrin.

 

 

In vitro knock-down activities against CD71-positive cells

Silencing activity was maintained after the conjugation. Compared to isotype control IgG Fab’ – siHPRT conjugate, anti-CD71 conjugate showed 1,000-10,000 fold boosting effect in HPRT silencing with mouse splenic B and T cells.
 

 

In vivo KD activities of anti-CD71 siHPRT (intravenous administration)

Regarding in vivo silencing, we dosed 10 mg/kg siHPRT conjugate intravenously and found the considerable silencing in muscular tissues on which CD71 are abundantly expressed.
 

 

1 month silencing with Single i.v. Injection

How about the duration of silencing? Here is an experiment where we have done a single intravenous injection looking at HPRT silencing and I think that you can see nice duration of silencing at 2, 3 and 4 weeks in the heart as well as the calf muscle. Negative control siRNA conjugate did not show the HPRT silencing.

 

Direct Application: Intramuscular Injection

In case of muscles, direct muscular injection would be attractive in terms of amount of siRNA dose. In previous page, 200 ug/mice was injected intravenously, however, only 1 ug muscular injection/leg showed considerable silencing. Forty ug injection was very potent and durable.

 
 

2. Evidence of Therapeutic Effect

In the following slides, we investigated if this platform can show therapeutic effect in a disease model. We selected very common disease to show the generality for muscular diseases.

Peripheral Artery Disease (PAD)

What is the effective treatment for PAD?

It is NOT limited to RNAi Therapy, so far few experimental reports have been found improving the running performance of PAD model mice.

 

PAD model mice and Study design

Here is a PAD model mice we are working with. We did Femoral artery ligation, this operation provides really nice model to investigate the efficacy of the drug, measuring the running performance by Treadmill.
So we tried local myostatin (MSTN) inhibition by intramuscular injection with anti-CD71 MSTN siRNA. We dosed 20 ug of siRNA conjugate / leg, 4 times weekly on PAD mice.

 

Phenotypic Changes in PAD mice

We looked at MSTN Expression and got very nice silencing in the left hands, and we did see significant increase in muscle weight by 17%, this is supported by histopathology as well, and most importantly, so far we’ve seen there are no successful articles on PAD mice, however we can see increase in running distance upon treatment of MSTN siRNA conjugate, giving us a good proof of concept of RNAi therapy using our platform. This is the first report to show the therapeutic effect in muscular disease with our proprietary technologies.

 

Summary

• Anti-CD71 antibody conjugates were applied for siRNA delivery to muscular tissues.
• Robust and Durable gene silencing has been observed in heart and skeletal muscle after systemic administration of siRNA conjugates.
• Intramuscular injection has also enabled robust and local gene silencing in gastrocnemius.
• This delivery platform will open new applications of siRNAs especially in muscular tissues.
• GenAhead Bio is looking forward to having a future of research and development partnership with pharmaceutical companies in this fields.