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Humanized Models

Genome edited animal models for studying your gene of interest

in-vivo-intro

“When people talk about ‘model organisms,’ many people think that the animal has to ‘model’ the human disease condition to have any impact .We argue that this is not the case. Simply studying what the homologous gene does in the context of the model organism can provide important insights into disease pathology. Rare genetic variants can be modeled in a number of different model organisms, informing causality between a particular variant and a pathogenic phenotype and offering clinicians and their patients hope for a diagnosis, insight into the mechanisms and pathways involved in a rare disease, and ways it might be treated.” Neff, E.P. Model matchmaking. Lab Anim 50, 39–42 (2021)

Using CRISPR and other genome editing methods, InVivo Biosystems can humanize or genetically engineer Zebrafish and C. elegans into powerful animal models for better understanding genetic mutations and disease biology discovery.

HElio Ortholog Gene Finder: find a usable model for studying your gene of interest.
Disease Gene Finder: Linking human disease genes and C. elegans genes and their corresponding phenotypes.

"InVivo Biosystems adapted to the needs of our project and created strains that introduced the variants of interest at the endogenous C.elegans locus.” In doing so, “they acted as a temporary post-doc, to speed this project along much faster than we could have done in the lab ourselves.” Ellen Gregory, PhD candidate, Starr & Luxton Labs, UC Davis. Read the customer story >>

Methods for Humanization

There are two types of humanizing changes can be made:

  • Whole gene humanization - swapping in human sequence for animal gene.
  • Point mutation humanization - inserting clinical variation into animal's gene.
Humanization with Point Mutation Humanization with Whole Gene
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Whole Gene Humanization

Whole gene humanization is often used to model the effects of the clinical variant.

  • The human coding sequence is used to replace the animal's coding sequence. 
  • CRISPR techniques are use to remove the entire coding sequence of the animals version of the gene.
  • A plasmid with homology flanked cargo is used to bring in the human coding sequence. 

We know we are looking at conserved biology between animal and human when the human gene rescues the function of the gene-deleted KO animal.  Next we put clinical variations into the humanized locus which allows us to examine any variation (rare or common) for its functional contribution to normal biology.  When we observe abnormal biology in a clinical variant, the system becomes a Human Avatar to screen drugs and find therapeutics that can restore normal function. 

We have found whole gene humanization can create a platform for highly translatable results in a model organism. The gene humanization method we use replaces the ortholog locus with human cDNA.  The cDNA is sequence-optimized for expression in the animal.

Gene-swap humanization technique.  Ortholog gene is removed from animal (KO) to determine if defective phenotype is present. Ortholog is replaced with human coding sequence to create gene-humanized animal.  Variants are installed in gene-humanized locus create Patient Avatars. Humanized animals in Patient Avatar format are phenotyped for activity defects.

Point Mutation Humanization

Point mutation humanization is a simpler, quicker and fairly effective - CRISPR techniques to replace a single amino acid with the suspect amino acid seen in the patient. The clinical variant seen in the patient is installed in the animal's version of the gene.

Note, this only works if sequence conservation is high and even then some variants that are pathogenic may not be at conserved positions. 

Model STXBP1a Patient Variants in C. elegans

In this example, the human coding sequence of STXBP1 was able to restore function when inserted as whole-gene humanization of the unc-18 locus. Referenced against a set of known pathogenic and benign variants, we were able to detect pathogenicity in a set of VUS.

Diagnostic curve for VUS assessment. (a) Transgene rescue demonstration by electrophysiology. (b) Transgene rescue demonstration by movement in liquid. (c) Deep phenotyping parameters for movement on solid surface comparis benign (green), pathogenic (red) and VUS (grey). d) diagnostic curve for pathogenicity assessment of VUS where values above harmonic mean indicate possible pathogenicity in the strain.

The video below shows that the human coding sequence of STXBP1 is inserted into the native locus of a C. elegans ortholog gene. The worms version of the gene is replaced with a human coding sequence.
- Left: the gene-swapped STXBP1 sequence functions and shows a significant level of activity.
- Middle: In contrast, the knock-out shows very little activity.
- Right: for the R406H genomic variant, its activity is somewhere in between the “humanized” and “knock-out” strain.

96 point mutations in 1 gene. All 96 mutations were created in the STXBP1 gene (which is associated with epilepsy in humans) via CRISPR. The worm homolog of STXBP1, unc-18, causes uncoordination and near-complete lack of pharyngeal pumping when knocked out. The functionality is restored by replacing the worm gene with the coding sequence for human STXBP1.

Model STXBP1a Patient Variants in Zebrafish

Illustration of a successful precise point mutation of Stxbp1a in Zebrafish. STXBP1a is a highly conserved zebrafish ortholog of human STXBP1 (87% identity). Using CRISPR/Cas9 technology, we were able to precisely generate a benign patient mutation at the conserved amino acid residue (CCC>CTG, p.P94L).

Who We Serve

We work with scientists seeking new or alternative ways to approach their research.

  • R&D researchers who need to test their hypotheses before moving to a mammalian system.
  • Pharmaceutical companies who need to quickly understand a compound’s mechanism of action as well as cytotoxic effect, CC50, IC50, SI, and etc.
  • Animal-conscious organizations who need to perform proof-of-concept validation.
  • Researchers studying longevity and senescence who want to eliminate the maintaining and handling of mouse model for months.
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