Yocardi: The new LNP is targeted to prepare CAR-T in vivo, and the anti-tumor effect is remarkable

October 20, 2022/eMedClub News/--CAR-T therapy, as a breakthrough medical technology, has brought hope to many patients with refractory hematological tumors, but at present, CAR-T therapy has shown certain limitations in the early clinical application process.
Among them, the complex in vitro preparation process and cytokine release syndrome (CRS) are two important obstacles
facing CAR-T therapy.

Recently, the Journal of Controlled Release published a paper entitled Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo, the corresponding author of which is Dr.
Yu Lei, founder and chief scientist of Youkadi
。 This study uses LNP delivery to achieve in vivo preparation of CAR-T therapy, which provides a potential solution
to the current clinical application dilemma of CAR-T therapy.
The researchers used CD3 antibody modified LNP to encapsulate short hairpin RNA (shRNA) containing human interleukin 6 (IL-6) and an integrated plasmid of CAR gene, which can produce CAR-T cells with missing IL-6 secretion function in mice through intravenous injection, and showed an anti-tumor effect comparable to conventional CAR-T treatment in mouse leukemia models, while reducing the release of IL-6 and reducing severeness The occurrence
of CRS.

This achievement not only represents an important breakthrough in the clinical application of CAR-T therapy, but also a blockbuster research achievement
in the field of in vivo CAR-T therapy.
The researchers believe that this technology improves the convenience of CAR-T therapy and helps to further promote it to clinical applications
.

At present, the CAR-T products on the market are all autologous CAR-T therapies, which need to undergo complex in vitro preparation processes and strict quality control, and each patient's CAR-T cells need to be individually customized, which cannot benefit more patients in one preparation, and the preparation process requires strict GMP cleanliness requirements
。 In addition, studies have shown that about 60-80% of patients treated with CAR-T will develop CRS, and its main mechanism is that CAR-T cells are activated after recognizing tumor cell antigens in the patient, continuously expanding and releasing a large number of cytokines, resulting in a systemic inflammatory response
.
Numerous studies suggest that elevated IL-6 is the leading cause of
CRS.
Therefore, the researchers in this paper overcome the current clinical application problems
by designing a CAR-T cell that can be prepared in vivo and has a loss of IL-6 secretion function.

The nanodelivery system delivers DNA to the body, making it possible
to generate CAR-T cells in vivo.
For example, in 2017, Nat Nano wrapped CAR DNA with cationic polymers and targeted delivery to T cells mediated by CD3 antibodies, directly transforming them into CAR-T cells, which provides a new protocol
for CAR-T therapy.
In this study led by Dr.
Yu Lei, the researchers used LNP as a gene delivery carrier, which has the advantages of druggability, high safety and strong durability compared with cationic polymers, and has been clinically verified
.

To achieve targeted delivery and transfection of CAR plasmids to T cells in vivo, the researchers prepared a CD3 antibody-modified cationic LNP vector containing an integrated plasmid containing shRNA containing IL-6 and the CAR gene, called AntiCD3-LNP/CAR19+shIL6
.
At the same time, the researchers used the method of adding microtubule-associated nuclear localization peptide (MTAS-NLS) to make the plasmid gene enter the nucleus to increase the transfection efficiency, and adding transposon to the plasmid gene made the nanoparticles have high transfection efficiency and durable and stable expression
.
After it was injected intravenously, the researchers evaluated the therapy in vitro function through cell killing experiments and the Cytokine secretion assay
.

The results of the study showed that AntiCD3-LNP/CAR19+shIL6 can edit T cells in vitro, target T cells in vivo and engineer them into CAR-T cells, and can also exert anti-tumor effects while reducing CRS
by modifying T cells.

Proper design of gene delivery systems is a key factor
affecting the efficient delivery and transfection of genes in vivo.
The safety of LNPs in educating T cells in vivo determines whether they can be used in the clinic, and the researchers used cationic lipid DOTAP and ionizable cationic lipid DLinDMA as the main carrier materials
for LNPs.
The material has a neutral charge around pH 7.
0, which makes LNPs stable in the blood, and its positive charge at low pH allows LNPs to escape from lysosomes
.
In addition, the high positive charge of LNPs may cause severe immune responses and toxic side effects
on normal tissues.
Therefore, the researchers optimized the formulation based on the previously loaded siRNA-loaded LNP to maintain the ZETTA potential of LNP at 0~2 mV
by adjusting the amounts of pDNA, DOTAP, and Dlin-MC3-DMA.

The transfection efficiency of LNP depends on the size of the pDNA, with smaller DNA fragments thought to be more easily encapsulated or easier to transfer to the
nucleus.
In order to ensure the high transfection efficiency of AntiCD3-LNP/CAR19+shIL6 in vivo, the researchers added IL-6 shRNA to the original CAR19 pDNA and deleted many unnecessary components, thereby reducing the length of the plasmid (5,314 bp in the AntiCD3-LNP/CAR 19 group, which is less than the classical CAR19).
8,504 bp) to promote lipid encapsulation; In addition, the researchers synthesized microtubule-associated nucleolocalization peptides (MTAS-NLS)-DSPE and added them to LNPs to facilitate LNPs to enter the nucleus while adsorbing and compressing plasmids
.

The researchers inserted the primordial transposon iPB7 into the plasmid and stably transfected T cells through LNPs, enabling T cells to stably express the CAR gene
in vivo.
Observations showed that T cell transduction lasted more than
90 days.
While the researchers successfully programmed T cells in vivo, off-target effects during treatment may reduce safety
.
The researchers found that some LNPs transfected macrophages in vivo and measured the number of CAR-expressing macrophages, and found that a small number of macrophages were transfected and expressed CAR, but these CAR-macrophages had difficulty proliferating
in vivo.
In contrast, CAR-T cells produced in the body can continue to proliferate
under the stimulation of tumor cells.
No significant toxicity
was observed by hematoxylin and eosin (H&E) staining of major organ sections.
This suggests that the expression of CAR in non-T cells does not cause significant damage
to other organs.

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Overall, the researchers found that programming T cells into CAR-T cells in vivo through LNP achieved an anti-tumor effect comparable to conventional CAR-T therapy, and reduced the risk
of CRS by knocking down IL-6.
This technology improves the convenience of using CAR-T technology and helps CAR-T therapy to further promote clinical applications
.

Resources:

1.
Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo

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