Page 55 - Annual report 2021-22

Annual Report 2021-22 |

Munia Ganguli

38

Genome editing using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-
Cas has thrown open a wide range of possibilities in gene correction to treat a large number of
diseases. One of the major challenges is the delivery of the gene editing machinery with high efficiency
and low toxicity. Some cargo formats like Ribonucleoprotein (RNP) complex and mRNA demand
carriers with specific attributes such as ensuring excellent cargo stability- owing to the high rate of
mRNA degradation and also efficiency in case of RNP complexes. Enabling excellent stability and
responsive release at the same time is a challenge.

Munia Ganguli’s lab is currently exploring a versatile biodegradable polymer system poly beta amino
ester (PBAE) for this purpose. Such polymers have the ability to condense nucleic acids into nano-sized
particles and also allow pH responsive release and degradation once uptaken by the target cells. They
are also highly amenable to modification, and the extent of modification can be flexibly controlled
allowing for a wide range of application-relevant functionalization.

The base polymer (DDS-90) was synthesized by stepwise Michael addition reaction with diacrylate and
amine at 1.2:1 ratio for 48hrs in DMF at 90°C under stirring. For M10 polymer (Mannitol doped
polymer), Mannitol diacrylate was doped at 10% to the diacrylate. The base polymers were capped
with a diamine group in THF for 2 hrs. The linear polymer DDS90 and modified polymer M10 were
complexed with a reporter mRNA (mCherry) in sodium acetate buffer. The DDS90 and M10 polymers
demonstrated stable complexation of mRNA and released the cargo when triggered by anionic
challenge. The nanoparticle size and charge were in the range of 100 nm as noted by Dynamic Light
Scattering and the nanocomplexes were spherical in shape as observed through Atomic Force
Microscopy. Both DDS90 and M10 exhibited excellent transfection of reporter mCherry(RFP) in HEK-
293 cell line.

Non-viral delivery of CRISPR-Cas9 components

In parallel, they have also rationally used ionic liquid BMIM[PF6] to design minimalistic non-viral
vectors for CRISPR-RNP delivery. Ionic liquid-RNP nanocomplexes were formed by non-covalent
interactions between the preformed RNP and the imidazolium-based ionic liquids. The complex
formation was optimized by screening for different weight ratios of Cas9 RNP to ionic liquids using
Atomic Force Microscopy. RNP remained structurally stable in BMIM[PF6] ionic liquid. The RNP-IL
particles remain as stable nanocomplexes up to 24 hours with no significant size change. The ionic
liquid-RNP nanocomplexes exhibited significant uptake by HEK-293 cells. The indicator protein was
seen as punctate fluorescence visible up to 24hrs post transfection. HEK cell lines constitutively
expressing GFP and TdTomato were transfected by BMIM[PF6]-RNP nanocomplexes with appropriate
sgRNA in independent experiments. The decrease of GFP or TdTomato signal is indicative of editing.
Similarly, nanocomplexes using M9 cell penetrating peptide and RNP complex have been prepared
and the surface charge, size and morphology have been studied using Dynamic Light Scattering and
Atomic Force Microscopy.