Developing Bottom-Up Induced Pluripotent Stem Cell Derived Solid Tumor Models Using Precision Genome Editing Technologies

Kelsie L Becklin^{1

2

3}, Garrett M Draper^{1

2

3}, Rebecca A Madden^{1

2

3}, Mitchell G Kluesner^{1

2

3}, Tomoyuki Koga^{4

5}, Miller Huang^{6

7}, William A Weiss^{8

9}, Logan G Spector^{1

2}, David A Largaespada^{1

2

3}, Branden S Moriarity^{1

2

3}, Beau R Webber^{1

2

3}

Affiliations

¹ Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
² Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
³ Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁴ Ludwig Cancer Research San Diego Branch, La Jolla, California, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁵ Department of Neurosurgery, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁶ Department of Pediatrics, University of Southern California, Los Angeles, California, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁷ Cancer and Blood Disease Institute, Children's Hospital Los Angeles and The Saban Research Institute, Los Angeles, California, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁸ Departments of Neurology, Pediatrics, Neurosurgery, Brain Tumor Research Center, and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA; and Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁹ Departments of Pediatrics, Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.

PMID: 35972367
PMCID: PMC9529369
DOI: 10.1089/crispr.2022.0032

Review

Developing Bottom-Up Induced Pluripotent Stem Cell Derived Solid Tumor Models Using Precision Genome Editing Technologies

Kelsie L Becklin et al. CRISPR J. 2022 Aug.

. 2022 Aug;5(4):517-535.

doi: 10.1089/crispr.2022.0032.

Authors

Affiliations

¹ Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
² Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
³ Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁴ Ludwig Cancer Research San Diego Branch, La Jolla, California, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁵ Department of Neurosurgery, University of Minnesota, Minneapolis, Minnesota, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁶ Department of Pediatrics, University of Southern California, Los Angeles, California, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁷ Cancer and Blood Disease Institute, Children's Hospital Los Angeles and The Saban Research Institute, Los Angeles, California, USA; Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁸ Departments of Neurology, Pediatrics, Neurosurgery, Brain Tumor Research Center, and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA; and Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.
⁹ Departments of Pediatrics, Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, San Francisco, California, USA.

PMID: 35972367
PMCID: PMC9529369
DOI: 10.1089/crispr.2022.0032

Abstract

Advances in genome and tissue engineering have spurred significant progress and opportunity for innovation in cancer modeling. Human induced pluripotent stem cells (iPSCs) are an established and powerful tool to study cellular processes in the context of disease-specific genetic backgrounds; however, their application to cancer has been limited by the resistance of many transformed cells to undergo successful reprogramming. Here, we review the status of human iPSC modeling of solid tumors in the context of genetic engineering, including how base and prime editing can be incorporated into "bottom-up" cancer modeling, a term we coined for iPSC-based cancer models using genetic engineering to induce transformation. This approach circumvents the need to reprogram cancer cells while allowing for dissection of the genetic mechanisms underlying transformation, progression, and metastasis with a high degree of precision and control. We also discuss the strengths and limitations of respective engineering approaches and outline experimental considerations for establishing future models.

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Conflict of interest statement

D.A.L. is the co-founder and co-owner of several biotechnology companies, including NeoClone Biotechnologies, Inc., Discovery Genomics, Inc. (recently acquired by Immusoft, Inc.), B-MoGen Biotechnologies, Inc. (recently acquired by Biotechne Corporation), and Luminary Therapeutics, Inc. D.A.L. holds equity in, serves as a Senior Scientific Advisor for and a Board of Director member for Recombinetics, a genome editing company. D.A.L. consults for Genentech, Inc., which is funding some of his research. W.A.W. is a co-founder of StemSynergy Therapeutics and holds equity in Nalo Therapeutics and Auron Therapeutics. The business of all these companies is unrelated to the contents of this article.

Figures

**FIG. 1.**
Bottom-up cancer modeling invokes the use of iPSC derived from somatic cells, which can then be differentiated into the cell-of-origin for a particular cancer alongside precise and timely genetic engineering to re-create cancer associated mutation/s. These models can be studied in many ways, including drug or genetic screens, used in organoid or mouse systems, or co-cultured with autologous or analogous immune cells to test new immunotherapies with the desired outcome of new therapeutics or new hypotheses. iPSC, induced pluripotent stem cell.

**FIG. 2.**
Precision genetic engineering tools. **(a)** CRISPR-Cas9 causes double-strand breaks when the sgRNA and DNA complement, activating the Cas9 enzyme. **(b)** CBE produces C → T transitions through the tethered cytidine deaminase to the nCas9 protein. **(c)** ABE produces A → G transitions via the tethered lab evolved DNA active adenosine deaminase to nCas9. **(d)** PE has a tethered RT to Cas9 and elongated sgRNA (pegRNA) such that the pegRNA is the template for the RT to incorporate into the DNA during repair. **(e)** CRISPRa/i has either an activator or a repressor tethered to a dCas9 to allow for site-specific gene activation or repression, respectively. **(f)** In many cases, an HR template will be desired for proper genetic engineering. These HR templates can be DNA plasmids, ssODNs, and AAV, among many other options. AAV, adeno-associated virus; ABE, Adenine base editor; CBE, Cytosine base editor; HR, homologous recombination; pegRNA, prime editor guide RNA; RT, reverse transcriptase; CRISPRa, CRISPR activation; CRISPRi, CRISPR interference; PE, prime editor; sgRNA, single guide RNA.

**FIG. 3.**
Common mutations found in cancer and suggested genetic engineering tools for induction.

See this image and copyright information in PMC

References

1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126(4):663–676; doi: 10.1016/j.cell.2006.07.024. - DOI - PubMed
1. Takahashi K, Tanabe K, Ohnuki M, et al. . Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861–872; doi: 10.1016/j.cell.2007.11.019. - DOI - PubMed
1. Brambrink T, Foreman R, Welstead GG, et al. . Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2008;2(2):151–159; doi: 10.1016/j.stem.2008.01.004. - DOI - PMC - PubMed
1. Maherali N, Sridharan R, Xie W, et al. . Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 2007;1(1):55–70; doi: 10.1016/j.stem.2007.05.014. - DOI - PubMed
1. Hu B-Y, Weick JP, Yu J, et al. . Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc Natl Acad Sci U S A 2010;107(9):4335–4340; doi: 10.1073/pnas.0910012107. - DOI - PMC - PubMed

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Developing Bottom-Up Induced Pluripotent Stem Cell Derived Solid Tumor Models Using Precision Genome Editing Technologies

Affiliations

Developing Bottom-Up Induced Pluripotent Stem Cell Derived Solid Tumor Models Using Precision Genome Editing Technologies

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Medical