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T Cells Instead of Viruses Used for CRISPR Gene Editing

posted by mrpg on Thursday July 12 2018, @10:22PM   Printer-friendly
from the WOPR dept.
Science

takyon writes:

T Cell Engineering Breakthrough Sidesteps Need for Viruses in CRISPR Gene-Editing

In an achievement that has significant implications for research, medicine, and industry, UC San Francisco scientists have genetically reprogrammed the human immune cells known as T cells without using viruses to insert DNA. The researchers said they expect their technique—a rapid, versatile, and economical approach employing CRISPR gene-editing technology—to be widely adopted in the burgeoning field of cell therapy, accelerating the development of new and safer treatments for cancer, autoimmunity, and other diseases, including rare inherited disorders.

The new method, described in the July 11, 2018 issue of Nature [DOI: 10.1038/s41586-018-0326-5] [DX], offers a robust molecular "cut and paste" system to rewrite genome sequences in human T cells. It relies on electroporation, a process in which an electrical field is applied to cells to make their membranes temporarily more permeable. After experimenting with thousands of variables over the course of a year, the UCSF researchers found that when certain quantities of T cells, DNA, and the CRISPR "scissors" are mixed together and then exposed to an appropriate electrical field, the T cells will take in these elements and integrate specified genetic sequences precisely at the site of a CRISPR-programmed cut in the genome.

[...] But just as important as the new technique's speed and ease of use, said Marson, also scientific director of biomedicine at the Innovative Genomics Institute, is that the approach makes it possible to insert substantial stretches of DNA into T cells, which can endow the cells with powerful new properties. Members of Marson's lab have had some success using electroporation and CRISPR to insert bits of genetic material into T cells, but until now, numerous attempts by many researchers to place long sequences of DNA into T cells had caused the cells to die, leading most to believe that large DNA sequences are excessively toxic to T cells.

To demonstrate the new method's versatility and power, the researchers used it to repair a disease-causing genetic mutation in T cells from children with a rare genetic form of autoimmunity, and also created customized T cells to seek and kill human melanoma cells.

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  • (Score: 1, Funny) by Anonymous Coward on Thursday July 12 2018, @10:51PM

    by Anonymous Coward on Thursday July 12 2018, @10:51PM (#706393)

    From the I-pity-the-fool dept.

  • (Score: 2) by Ken_g6 on Thursday July 12 2018, @11:07PM (3 children)

    by Ken_g6 (3706) on Thursday July 12 2018, @11:07PM (#706397)

    Shouldn't it be "CRISPR Instead of Viruses Used for T Cell Gene Editing"?

    • (Score: 1, Funny) by Anonymous Coward on Thursday July 12 2018, @11:18PM

      by Anonymous Coward on Thursday July 12 2018, @11:18PM (#706399)

      T Virus Instead of Cells Used for CRISPR Gene Editing

    • (Score: 0) by Anonymous Coward on Friday July 13 2018, @04:23AM (1 child)

      by Anonymous Coward on Friday July 13 2018, @04:23AM (#706516)

      No, they report using electroporation instead of viruses to get long dna sequences in there while killing fewer cells than usually seen with this method. The DNA sequences are then used with crispr. I checked out the first experiment they show:

      Contrary to expectations, we found that co-electroporation of human primary T cells with CRISPR–Cas9 ribonucleoprotein (RNP)10,11 complexes and long (>1 kb) linear dsDNA templates reduced the toxicity associated with the dsDNA template (Extended Data Fig. 1a–e). Cas9 RNPs were co-electroporated with a dsDNA HDR template designed to introduce an N-terminal green fluorescent protein (GFP) fusion in the housekeeping gene RAB11A (Fig. 1a). Both cell viability and the efficiency of this approach were optimized by systematic exploration (Fig. 1b and Extended Data Fig. 1f–h), resulting in GFP expression in up to 50% of primary human CD4+ and CD8+ T cells.
      [...]
      Efficiency and viability were measured 4 days after electroporation
      [...]
      Immediately after isolation, T cells were stimulated for 2 days with anti-human CD3/CD28 magnetic dynabeads
      [...]
      After electroporation, T cells were cultured in media with IL-2 at 500 U ml−1. Throughout the culture period T cells were maintained at an approximate density of 1 million cells per ml of media. Every 2–3 days after electroporation, additional media was added, along with additional fresh IL-2 to bring the final concentration to 500 U ml−1, and cells were transferred to larger culture vessels as necessary to maintain a density of 1 million cells per ml.
      [...]
      RNPs and HDR templates were electroporated 2 days after initial T cell stimulation. T cells were collected from their culture vessels and magnetic anti-CD3/anti-CD28 dynabeads were removed by placing cells on an EasySep cell separation magnet for 2 min. Immediately before electroporation, de-beaded cells were centrifuged for 10 min at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μl buffer per 1 million cells. For optimal editing, 1 million T cells were electroporated per well using a Lonza 4D 96-well electroporation system with pulse code EH115.
      [...]
      Except where noted otherwise, ‘viability’ refers to the number of live cells in an experimental condition (expressed as a percentage) relative to an equivalent population that went through all protocol steps except for the actual electroporation (no electroporation control). ‘Efficiency’ refers to the percentage of live cells in a culture expressing the ‘knocked-in’ exogenous sequence (such as GFP). Finally, the total number of cells positive for the desired modification was calculated by multiplying the efficiency by the absolute cell count.
      [...]
      Arrayed testing of electroporation pulse conditions showed that, in general, conditions yielding higher HDR efficiency decreased viability.

      https://www.nature.com/articles/s41586-018-0326-5 [nature.com]

      Extended data figure 1 a/h clearly shows that the more cells they kill, the higher the percentage of mutants they see. They started with 1 million cells, killed about 60% by day 2 (to 400k cells), and this grew back to 1 million by day for at which time about 30% of those remaining met their criteria for success (some threshold amount of GFP detected).

      Also, apparently T-cells can divide about 12 times a day (this is really fast for human cells and probably explains why so much of this "editing" work is done with T-cells):

      In conclusion, we have demonstrated that CD8+ T cells responding to an antigenic stimulus can undergo extremely rapid proliferation with cell cycle times of as short as 2 hrs.

      http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015423 [plos.org]

      So, we need to get 300k cells by 4 days when they divide 12 times per day. How many initial mutant cells are required to do this? The answer is you can get that many within 2 days from a single original mutant cell:

      n0        = 1
      divPerDay = 12
      nDays     = 0:4
      nDiv      = divPerHr*nDays
      nCell     = n0*2^nDiv

      > cbind(nDays, nDiv, nCell)
           nDays nDiv        nCell
      [1,]     0    0 1.000000e+00
      [2,]     1   12 4.096000e+03
      [3,]     2   24 1.677722e+07
      [4,]     3   36 6.871948e+10
      [5,]     4   48 2.814750e+14

      So how many cells actually got "edited" here? How fast were these cells proliferating under these exact conditions. You can see the division rate may be far less and still explain this data. Also figure 1c shows 0.1% to 0.2% of control cells met their criteria for success. This works out to 1000 initial cells, in that case you can get ~250k cells after 4 days by only dividing twice per day.

      • (Score: 0) by Anonymous Coward on Friday July 13 2018, @04:25AM

        by Anonymous Coward on Friday July 13 2018, @04:25AM (#706518)

        Damn, typo in the code (I switched from divPerHr to divPerDay so the table wasnt huge, the results are correct though):

            n0        = 1
            divPerDay = 12
            nDays     = 0:4
            nDiv      = divPerDay*nDays
            nCell     = n0*2^nDiv

            > cbind(nDays, nDiv, nCell)
                 nDays nDiv        nCell
            [1,]     0    0 1.000000e+00
            [2,]     1   12 4.096000e+03
            [3,]     2   24 1.677722e+07
            [4,]     3   36 6.871948e+10
            [5,]     4   48 2.814750e+14

  • (Score: 1) by Skwearl on Thursday July 12 2018, @11:34PM (1 child)

    by Skwearl (4314) Subscriber Badge on Thursday July 12 2018, @11:34PM (#706405)

    Zombies. This is how you get zombies. Didn't these scientists watch the 40 years of B movies?

    • (Score: 2) by bitstream on Friday July 13 2018, @12:00AM

      by bitstream (6144) on Friday July 13 2018, @12:00AM (#706416) Journal

      They took part in the "No scientist left behind" program delivered by the department of diversity.

  • (Score: 2) by Snotnose on Friday July 13 2018, @03:48AM (1 child)

    by Snotnose (1623) on Friday July 13 2018, @03:48AM (#706498)

    When I was in my 20s I thought medical science would have advanced enough by now so that living past 100 would be the norm, Now it looks like I'm gonna miss that by a decade (as in, I'll die in my 70s 10 years before 100+ is normal).

    I figure the day I wake up without any aches or pains is the day I died in my sleep.

    / In my 20s I thought organ transplants would do the job
    // DNA editing wasn't even on the radar

    --
    When the dust settled America realized it was saved by a porn star.
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