A CRISPR super repressor enhances gene therapy

SUper CRISPR repressor enhance gen therapy

Date: 8th September 2020

Gene therapy seeks to modify or manipulate the expression of a gene or to alter the biological properties of living cells for therapeutic use.  This generally relies on viruses, such as adeno-associated virus (AAV), to deliver genes into a cell however, these viruses can elicit an immune response and can render the treatment ineffective.  Now scientists, use a CRISPR-based synthetic repressor to modulate the immune system which subsequently enhances AAV-delivered gene therapy.

Transient modulation of the immune system genes, without engineering a permanent change in the DNA code, could be an effective strategy to modulate many inflammatory conditions and could be used as an immune modulator to create more effective gene therapies.

Currently, many gene therapies fail due to patients having a pre-existing immunity to the viruses used as the delivery vehicles, or an initial AAV gene therapy elicits an immune response such that the therapy can’t be re-administered.

Now scientists from the University of Pittsburgh, US, have created a CRISPR-based system that can effectively prevent immunity to viral vectors in mice – reprogramming immune homeostasis, and able to prevent or treat sepsis.  Furthermore, the system also improved the efficiency of a subsequent AAV/CRISPR treatment for repression of a proprotein involved in the regulation of cholesterol homeostasis, lowering cholesterol levels in the blood.

‘Super’ repressor system

To start the team harnessed the power of the CRISPR-Cas system to create an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis.  They used two transcriptional repressors—heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB)—which were fused to a bacteriophage MS2 coat protein.  The coat protein then bound to small RNA hairpins, here gRNA aptamers, on the nuclease competent CRISPR complex – creating a ‘super’ repressor.

But which target could modulate the immune system?  The team chose Myeloid differentiation primary response 88 (MyD88), which is a key node in the innate and adaptive immune response.

Primary experiments showed that the gRNAs (designed to target MyD88) and the engineered MS2 packaged into two separate AAVs (together called AAV/Myd88) and delivered into Cas9 nuclease transgenic mice showed a significant repression of the endogenous Myd88.  This repression ranged from ~63-84% reduction in expression levels in a variety of tissues.  In contrast, AAV delivery without the gRNAs led to an increase in Myd88, which confirmed these AAVs can elicit immune pathways in host animals.

Antibody response

Next the team wanted to assess the antibody response to AAVs in their system.  They, measured the IgG response against the AAVs three weeks after injection into the transgenic mice, and detected a 50% decrease in plasma IgG2a levels against the AAV in animals treated with the super repressor system compared with the control animals.  This indicated that the treatment could modulate the immune system, suppressing antibody formation against the therapy.

Antibody formation against AAVs is an often a barrier to re-administration of AAV-based gene therapies.  The team therefore wanted to assess whether the repressor system could overcome this limitation.  Indeed, pre-treatment with AAV/Myd88 and subsequent treatment 7 days later with AAVs carrying either a reporter gene (LacZ) or Cas9 showed higher transcript levels of LacZ in the blood, and lower antibody levels to Cas9 than the mock treated animals.  With these promising results showing enhanced activity of the second AAV treatment with AAV/Myd88 pre-treatment, the team wanted to apply the system to altering the gene expression in vivo of a potential therapeutic target.

gene therapy

Gene therapy potential

To address this, the researchers administered to mice two sequential rounds of AAV-based gene therapies (a week apart) after the animals had received the AAV/Myd88 pre-treatment.  This time the AAV carried a cassette for CRISPR-mediated repression of proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 encodes an enzyme which initiates ingestion of the low-density lipoprotein (LDL) receptors, therefore repression of this gene should lower blood LDL-cholesterol levels by elevating the presence of the receptors.  In this experiment the team observed lower antibodies to the AAVs in the pre-treated animals compared with the control animals, which was accompanied by better PCSK9 repression and lower plasma cholesterol levels, suggesting increased efficiency of the gene therapies.

But would the technology work to control an inflammatory disease?  To test this the team applied the system to Cas9-transgenic mice – AAV/Myd88 pre-treatment were performed then 3 weeks later septicaemia was induced.   During the analysis the team saw systemic markers associated with septicaemia and tissue damage were significantly reduced, and that a wide range of inflammatory and immune-related cytokines that were directly or indirectly downstream of Myd88 signalling were not upregulated as was seen in the control animals. Importantly, similar results were seen when the system was applied to wild type mice, here the Cas9 cassette was delivered by an additional AAV.

Lastly, as a proof-of-concept in a therapeutic setting nanoparticles were used to carry DNA encoding Myd88-targeting CRISPR, here the team showed that the repressor system was able to confer protection against septicaemia in mice.

Conclusion and future applications

The team here have created a transcriptional therapeutic module for synthetic control of the immune response using a newly developed CRISPR-based transcriptional super-repressor against endogenous Myd88. They show the system is effective at modulating downstream immune signalling and can create a protective phenotype in vivo.

The system offers us an exciting advancement in enhancing gene therapies, by suppressing immunoglobin production to AAVs, it may help us to circumvent challenges that are commonly faced with AAV-based clinical gene therapies and acquired immunity.

However, as with any potential therapy translating into the clinical, a vigorous safety and efficacy trial will be required. Although, initial data indicated that at least in mice, CRISPR-mediated Myd88 repression did not create adverse long term effects.

The strategy was also effective in modulating the systemic inflammatory response.  This could be an interesting approach for treating sepsis and other such inflammatory diseases such as COVID-19.  Here, poor outcomes have been associated with cytokine storms in patients with severe symptoms.

With this in mind we have recently seen that sequestering of disease-inducing molecules is becoming a growing area of interest for translation research.  We recently reported the development of cellular nanosponges as an effective medical countermeasure to SARS-CoV-2 which can attract, sequester and neutralise the virus in vitro.   Furthermore, these nanosponges may also sequester inflammatory cytokines, which cause many of the most lethal manifestations of COVID-19. Scientists have also developed a nanotrap technology to efficiently capture and attenuate a group of inflammatory mediators for effective sepsis treatment. However, whilst these therapies prove effective at removing inflammatory molecules that have already been expressed, repression of Myd88 via the super repressor may prevent their accumulation in the first place.  How this field evolves, and which approach proves most successful for treating inflammatory conditions is yet to unfold.


For more information please see the press release from the University of Pittsburgh Medical Center

Moghadam, F., R. LeGraw, J. J. Velazquez, N. C. Yeo, C. Xu, J. Park, A. Chavez, M. R. Ebrahimkhani and S. Kiani (2020). “Synthetic immunomodulation with a CRISPR super-repressor in vivo.” Nature Cell Biology 22(9): 1143-1154.