HIV DNA circularizes to circumvent CRISPR-based treatments


CRISPR-Cas9 technology shows promise for treating inherited disorders, but scientists are also exploring its usefulness for excising embedded viruses. Early reports have demonstrated that CRISPR effectively excises the integrated human immunodeficiency virus type 1 (HIV) from cells.1.2 For Michele Lai of the University of Pisa, these studies raised an important question: what happens to the excised DNA?

He set out to answer this question and recently published in the Journal of Virology that some of the excised viral DNA molecules form stable DNA circles that can reintegrate into the genome.3 This phenomenon can pose a challenge to those working on CRISPR-based HIV treatments.

“Even if CRISPR works in 100% of the T cells, you would still have a good number of cells where the [plasmids] may restart the infection. —Mauro Pistello, University of Pisa

“In this context of genomic instability, the cell does not understand what the DNA of the cell is and what the virus is. I hypothesized that DNA repair mechanisms might create a circle of viral DNA that is more resistant to [degradation]“, said Lai.

To test this idea, Lai and his colleagues treated HIV-infected cells with CRISPR and used a technique to specifically amplify circular viral DNA. This allowed them to detect tiny plasmids carrying viral sequences in CRISPR-treated cells.

“We did some calculations, and there was about one cell in 1,000,” said Mauro Pistello, lead author of the paper. “Even if CRISPR works in 100% of the T cells, you would still have a good number of cells where the [plasmids] may restart the infection.

When the researchers sequenced the viral plasmids, they found that almost all of them contained an intact LTR. The LTR serves as an HIV promoter to activate transcription,2 so Lai wondered if viral plasmids could allow a cell to produce viral proteins.

To find out, Lai mixed the plasmids with HIV proteins that transcribe the viral genome and found that they did form infectious virions, but at a low rate.3

Michele Lai discovered that HIV forms infectious DNA plasmids after CRISPR processing.

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“The idea that even if you have successful gene therapy excision of the virus, it can reassemble into something functional is a very provocative scenario that should be further investigated in primary cells or animal models,” he said. Satish Pillai of the Vitalant Research Institute. and the University of California, San Francisco (UCSF), which did not participate in the study. “Understanding the fate of excision products and the cells that harbor these excision products will be essential to designing a functional and safe gene therapy strategy in the future.”

As the development of this strategy progresses, Lai and his co-authors propose revising CRISPR approaches for cutting the HIV genome into multiple fragments. “The idea of ​​a multi-targeting strategy to combat HIV with gene therapy now has two levels of support: discouraging the evolution of viral resistance and greatly reducing, if not eliminating, the possibility that the virus could reassemble into a functional molecule,” Pillai said.

However, this solution reduces the effectiveness of gene therapy and increases the chances of cutting unintended genomic locations.7.8 As they move forward, Lai, Pistello and collaborators will continue to refine their strategy to strike a balance between avoiding viral reassembly and maintaining host cell genomic integrity in pursuit of gene therapy. safe and effective for HIV.

The references

  1. H. Ebina et al., “Harnessing the CRISPR/Cas9 system to disrupt the latent HIV-1 provirus”, Scientific representative3:2510, 2013.
  2. PK Dash et al., “Sequential LASER ART and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice”, Nat Comm10(1):2753, 2019.
  3. M. Lai et al., “CRISPR/Cas9 ablation of integrated HIV-1 accumulates proviral DNA circles with reshaped long terminal repeats,” J. Virol95(23):e0135821, 2021.
  4. ME Barry et al., “Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection,” Hum Gene Ther10(15):2461-80, 1999.
  5. M. Kosicki et al., “Repair of CRISPR-Cas9-induced double-strand breaks leads to large deletions and complex rearrangements”, Nat Biotechnol, 36(8): 765-71, 2018.
  6. G. Magro et al., “Targeting and understanding HIV latency: the CRISPR system against the provirus”, Pathogens, 10(10): 1257, 2021.
  7. “CRISPR Guide”, Addgene, https://www.addgene.org/guides/crispr/
  8. M. Naeem et al., “Latest strategies developed to minimize off-target effects in CRISPR-Cas-mediated genome editing”, cells9(7): 1608, 2020.

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