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Use of CRISPR/CAS9 to Edit Genome of Pigs: Recominetics announces $10M Funding Round, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
Gene-editing startup raising $10M to expand staff
Nov 25, 2015
Katharine Grayson
Staff reporter
Minneapolis / St. Paul Business Journal
Recombinetics Inc. is seeking $10 million in funding as it ramps up sales of its genetically tweaked animals.
The St. Paul-based biotech company’s recent round has already brought in about about $2.8 million from friends and family, said Chief Operating Officer Kyle Dawley. Company officials hope to close out the round within the next two months and add about 10 employees to its staff of 25.
Recombinetics edits pigs’ genes for biomedical research purposes. Photo source: Simone Van Den Berg
Recombinetics uses gene-editing technology to tweak animals for the agribusiness and biomedical markets. It’s biomedical business centers around pigs, which the company modifies for research purposes. That side of the company’s business already generates revenue, Dawley said, though he declined to reveal sales figures.
The company focuses on pigs, touting them as better research subjects than mice when it comes to testing medical devices and drugs for use in humans.
“Pigs are — size-wise and genetically — a lot more like humans than rats and mice,” Dawley said.
One of Recombinetics’ long-term goals is grow human organs inside pigs.
The company aims to modify livestock for food consumption as well. One of its projects calls for creating hornless cattle by taking a gene from one breed and putting into another.
FDA Guidance On Source Animal, Product, Preclinical and Clinical Issues Concerning the Use of Xenotranspantation Products in Humans – Implications for 3D BioPrinting of Regenerative Tissue
Reporter: Stephen J. Williams, Ph.D.
The FDA has submitted Final Guidance on use xeno-transplanted animal tissue, products, and cells into human and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.
This document is to provide guidance on the production, testing and evaluation of products intended for use in xenotransplantation. The guidance includes scientific questions that should be addressed by sponsors during protocol development and during the preparation of submissions to the Food and Drug Administration (FDA), e.g., Investigational New Drug Application (IND) and Biologics License Application (BLA). This guidance document finalizes the draft guidance of the same title dated February 2001.
For the purpose of this document, xenotransplantation refers to any procedure that involves the transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs. For the purpose of this document, xenotransplantation products include live cells, tissues or organs used in xenotransplantation. (See Definitions in section I.C.)
This document presents issues that should be considered in addressing the safety of viable materials obtained from animal sources and intended for clinical use in humans. The potential threat to both human and animal welfare from zoonotic or other infectious agents warrants careful characterization of animal sources of cells, tissues, and organs. This document addresses issues such as the characterization of source animals, source animal husbandry practices, characterization of xenotransplantation products, considerations for the xenotransplantation product manufacturing facility, appropriate preclinical models for xenotransplantation protocols, and monitoring of recipients of xenotransplantation products. This document recommends specific practices intended to prevent the introduction and spread of infectious agents of animal origin into the human population. FDA expects that new methods proposed by sponsors to address specific issues will be scientifically rigorous and that sufficient data will be presented to justify their use.
Examples of procedures involving xenotransplantation products include:
transplantation of xenogeneic hearts, kidneys, or pancreatic tissue to treat organ failure,
implantation of neural cells to ameliorate neurological degenerative diseases,
administration of human cells previously cultured ex vivo with live nonhuman animal antigen-presenting or feeder cells, and
extracorporeal perfusion of a patient’s blood or blood component perfused through an intact animal organ or isolated cells contained in a device to treat liver failure.
Imagine stripping out the failing components of an old car — the engine, transmission, exhaust system and all of those parts — leaving just the old body and other structural elements. Replace those old mechanical parts with a brand new electric, hydrogen powered, biofuel, nuclear or whatever kind of engine you want and now you have a brand new car. It has an old frame, but that’s okay. The frame wasn’t causing the problem, and it can live on for years, undamaged.
When challenged to design internal organs, tissue engineers are taking a similar approach, particularly with the most complex organs, like the heart, liver and kidneys. These organs have three dimensional structures that are elaborate, not just at the gross anatomic level, but in microscopic anatomy too. Some day, their complex connective tissue scaffolding, the stroma, might be synthesized from the needed collagen proteins with advanced 3-D printing. But biomedical engineering is not there yet, so right now the best candidate for organ scaffolding comes from one of humanity’s favorite farm animals: the pig.
Chimera alarmists connecting with anti-biotechnology movements might cringe at the thought of building new human organs starting with pig tissue, but if you’re using only the organ scaffolding and building a working organ from there, pig organs may actually be more desirable than those donated by humans.
How big is the anti-chimerite movement?
Unlike anti-GMO and anti-vaccination activists, there really aren’t too many anti-chemerites around. Nevertheless, there is a presence on the web of people who express concern about mixing of humans and non-human animals. Presently, much of their concern is focussed on the growing of human organs inside non-human animals, pigs included. One anti-chemerite has written that it could be a problem for the following reason:
Once a human organ is grown inside a pig, that pig is no longer fully a pig. And without a doubt, that organ will no longer be a fully human organ after it is grown inside the pig. Those receiving those organs will be allowing human-animal hybrid organs to be implanted into them. Most people would be absolutely shocked to learn some of the things that are currently being done in the name of science.
The blog goes on to express alarm about the use of human genes in rice and from there morphs into an off the shelf garden variety anti-GMO tirade, though with an an anti-chemeric current running through it. The concern about making pigs a little bit human and humans a little bit pig becomes a concern about making rice a little bit human. But the concern about fusing tissues and genes of humans and other species does not fit with the trend in modern medicine.
Utilization of pig tissue enters a new age
A porcine human ear for xenotransplantation. source: The Scientist
For decades, pig, bovine and other non-human tissues have been used in medicine. People are walking around with pig and cow heart valves. Diabetics used to get a lot of insulin from pigs and cows, although today, thanks to genetic engineering, they’re getting human insulin produced by microorganisms modified genetically to make human insulin, which is safer and more effective.
When it comes to building new organs from old ones, however, pig organs could actually be superior for a couple of reasons. For one thing, there’s no availability problem with pigs. Their hearts and other organs also have all of the crucial components of the extracellular matrix that makes up an organ’s scaffolding. But unlike human organs, the pig organs don’t tend to carry or transfer human diseases. That is a major advantage that makes them ideal starting material. Plus there is another advantage: typically, the hearts of human cadavers are damaged, either because heart disease is what killed the human owner or because resuscitation efforts aimed at restarting the heart of a dying person using electrical jolts and powerful drugs.
Rebuilding an old organ into a new one
How then does the process work? Whether starting with a donated human or pig organ, there are several possible methods. But what they all have in common is that only the scaffolding of the original organ is retained. Just like the engine and transmission of the old car, the working tissue is removed, usually using detergents. One promising technique that has been applied to engineer new hearts is being tested by researchers at the University of Pittsburgh. Detergents pumped into the aorta attached to a donated heart (donated by a human cadaver, or pig or cow). The pressure keeps the aortic valve closed, so the detergents to into the coronary arteries and through the myocardial (heart muscle) and endocardial (lining over the muscle inside the heart chambers) tissue, which thus gets dissolved over the course of days. What’s left is just the stroma tissue, forming a scaffold. But that scaffold has signaling factors that enable embryonic stem cells, or specially programed adult pleuripotent cells to become all of the needed cells for a new heart.
Eventually, 3-D printing technology may reach the point when no donated scaffolding is needed, but that’s not the case quite yet, plus with a pig scaffolding all of the needed signaling factors are there and they work just as well as those in a human heart scaffold. All of this can lead to a scenario, possibly very soon, in which organs are made using off-the-self scaffolding from pig organs, ready to produce a custom-made heart using stem or other cells donated by new organ’s recipient.
David Warmflash is an astrobiologist, physician, and science writer. Follow @CosmicEvolution to read what he is saying on Twitter.
And a Great Article in The Scientist by Dr. Ed Yong Entitled
To cope with a growing shortage of hearts, livers, and lungs suitable for transplant, some scientists are genetically engineering pigs, while others are growing organs in the lab.
.. where Joseph Vacanti and David Cooper figured that using
“engineered pigs without the a-1,3-galactosyltransferase gene that produces the a-gal residues. In addition, the pigs carry human cell-membrane proteins such as CD55 and CD46 that prevent the host’s complement system from assembling and attacking the foreign cells”
… thereby limiting rejection of the xenotransplated tissue.
In addition to issues related to animal virus transmission the issue of optimal scaffolds for organs as well as the advantages which 3D Printing would have in mass production of organs is discussed:
To Vacanti, artificial scaffolds are the future of organ engineering, and the only way in which organs for transplantation could be mass-produced. “You should be able to make them on demand, with low-cost materials and manufacturing technologies,” he says. That is relatively simple for organs like tracheas or bladders, which are just hollow tubes or sacs. Even though it is far more difficult for the lung or liver, which have complicated structures, Vacanti thinks it will be possible to simulate their architecture with computer models, and fabricate them with modern printing technology. (See “3-D Printing,” The Scientist, July 2012.) “They obey very ordered rules, so you can reduce it down to a series of algorithms, which can help you design them,” he says. But Taylor says that even if the architecture is correct, the scaffold would still need to contain the right surface molecules to guide the growth of any added cells. “It seems a bit of an overkill when nature has already done the work for us,” she says.
Other articles of FDA Guidance and 3D Bio Printing on this Open Access Journal Include:
The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research
Updated 6/25/2019
Writer. Reporter: Stephen J. Williams, Ph.D.
The choice of suitable animal model of disease may define future success or failure for drug development, basic and translational research, or biomarker discovery projects. Indeed, as highlighted in one of my earlier posts “Heroes in Medical Research: Developing Models for Cancer Research”, the choice of animal to model a human disease can have drastic implications in the basic researchers ability to understand metabolic and genetic factors causally associated with disease development. As described in that post the King rat model led to our understanding of the genetics of early development and sex determination while early mouse models helped us to understand the impact of microenvironment on cell fate and the discovery of stem cells. In addition, transgenic and immunodeficient mice resulted in transformational studies on our understanding of cancer. Small rodent models are ideal for following reasons:
Ease of genetic manipulation
Availability of well-defined models
Ease of low cost of use
Regardless of these benefits many investigators in industry and academia are looking to models of human disease in animals more closely resembling human anatomy, physiology, and genetics.
There is a growing need for alternative animal models in cancer research.
Similar anatomic dimensions: i.e. Adult Gottingen minipigs are 70kg (adult human male weight)
Similar organ size and structure to humans organ size and structure
Pig genome sequencing project nearly complete
Ability to manipulate pig genetics
The post had discussed the development of a severe combined immunodeficient (SCID) pig by investigators at Iowa State and Kansas State University. This line of pigs, selected on a specific diet, could act as recipients for human cancer cell lines, a proof of their SCID phenotype.
A report featured on Fierce Biotech Research “MU Scientists Successfully Transplant, Grow Stem Cells in Pigs” discussed the development of a new genetically-modified immunodeficient porcine model by researchers at the University of Missouri, recently published in Proceedings of the National Academy of Sciences[1].
These pigs are available from the National Swine Resource and Research Center (http://nsrrc.missouri.edu).
For the report on Fierce Biotech Research please follow the link below:
The report in FierceBiotech highlights the type of studies an immunocompromised pig model would be useful for including:
Regenerative medicine
Xenotransplantation
Tumor growth and efficacy studies
Comments in the post from the investigators explained the benefits of developing such a porcine model system including:
“The rejection of transplants and grafts by host bodies is a huge hurdle for medical researchers,” said R. Michael Roberts, Curators Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center. “By establishing that these pigs will support transplants without the fear of rejection, we can move stem cell therapy research forward at a quicker pace.”
The studies main investigators, Drs. Randall Prather and R. Michael Roberts, both of University of Missouri, along with first authors Kiho Lee, Deug-Nam Kwon and Toshihiko Ezashi, used biallellic mutation of the RAG2 gene in Gottingen minipig fibroblasts and then subsequent somatic cell nuclear transfer (SCNT) to produce the RAG2-/- animals. (Rag2 is a protein involved in V(D)J recombination of antibodies during early B and T cell development. See GeneCard link above)
As proof of their SCID phenotype the authors showed that
these RAG2-/- animals could act as host for human induced pluripotent stem cells
act as recipient for allogeneic porcine stem cells
reduced levels of (CD21+) B cells and (CD3+) T cells
growth retardation if housed under standard, non-sterile conditions
Details of the study are given below:
Methodology Used
For Production of Gottingen minipigs carrying the RAG2 mutation
To produce targeted mutations in RAG2:
TALENS () were constructed to produced mutation in exon 2 of RAG2
Constructed TALENS and reporter electroporated in fetal-derived pig fibroblasts
SCNT used to transfer RAG2 mutant nuclei to donor oocytes
9 embryo transfers resulted in 22 live piglets
Piglets genotyped as either monoallelic or biallelic RAG2 mutant
RAG2wild-type and mutants housed in either pathogen-free or normal housing conditions
To verify SCID phenotype of litter by either
Graft acceptance of human iPSCs and teratoma formation
– Fibroblasts from human umbilical cord reprogrammed to pluripotency; verified by pluripotent markers POUSF1, NANOG, SSEA-3)
– Two human and porcine iPSC lines with trophoblastic properties[2] were injected subcutaneously in ear or flank
– Tumor formation analyzed by immunohistochemistry using markers:
– Analysis of piglet spleen cells for B cell population (CD21)
– Analysis of piglet spleen cell for T cell population (CD3)
C. Histology
– histo evaluation of thymus, spleen
– marker evaluation of spleen using anti-CD79A (B cells), CD3 (T cells),
CD335 (NK cells)
Results
TALEN produced a variety of indels (insertion/deletions) and three RAG2 mutatnt colonies (containing monoallelic, mix of mono and biallelic) used for SCNT.
Three litters produced 16 piglets (eight survived [four mono and four biallelic]
Biallelic RAG2 mutants showed slower weight gain than wild type or monoallelic mutants with signs of inflammation and apoptosis in spleen and designated “failure to thrive” in standard housing…needed a clean environment to thrive.
Biallelic mutant pigs lacked mature CD21 B cells and CD3 T cells but contained macrophages and NK cells.
Implantation of human and allogenic porcine pluripotent stem cells (trophoblastic) showed rapid development of teratomas. References
Ezashi T, Matsuyama H, Telugu BP, Roberts RM: Generation of colonies of induced trophoblast cells during standard reprogramming of porcine fibroblasts to induced pluripotent stem cells. Biology of reproduction 2011, 85(4):779-787.
Updated 6/25/2019
The following articles represent an update on the ability to create genetically predisposed porcine models of cancer. The ability to utilize transposable elements to introduce genetic changes in porcine cells in combination with Somatic Cell Nuclear Transfer technology with the ultimate goal to create a transgenic minipig is discussed. The next two articles describe the ability of the scid pig to act as a recipient for human ovarian cancer cells and description of a transgenic inducible porcine intestinal tumor model.
Transgenic Res. 2011 Jun;20(3):533-45. doi: 10.1007/s11248-010-9438-x. Epub 2010 Aug 29.
Pig transgenesis by Sleeping Beauty DNA transposition.
Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.
This paper makes use of two technologies: transposon mediated gene transfer to introduce foreign DNA, for example a disease predisposition gene, into oocytes or early embryos, without the use of viral vectors; and use of SCNT to clone a minipig from viable embryos.
The transposon mediated system is based on the Sleeping Beauty (SB) vector system, which is a cut and paste DNA transposon belonging to the Tc1/mariner superfamily of transposable elements(1). Transposable DNA elements are mobile genetic elements which integrate into genomic DNA, in the case of the SB system into discrete sequence elements of actively transcribed genes. The system consists of two entities: 1) a transposase responsible for cutting and pasting the mobile element and 2) the transposon; the vectorial DNA sequence which is inserted into genomic DNA. SB transposition has been used to integrate exogenous genetic elements into the genome of various mammalian species(2) and to make tumor models in mice (3-7) and to transform, ex-vivo, porcine ovarian epithelial cells (8) and to stably integrate GFP containing vectors into porcine fibroblast genome(9). Because of the efficiency and nonviral integration of exogenous vectors into mammalian systems, Sleeping Beauty system has been considered as a potential therapeutic gene transfer modality (10-12).
Li, Z.H., Liu, D.P., Wang, J., Guo, Z.C., Yin, W.X., and Liang, C.C. Inversion and transposition of Tc1 transposon of C. elegans in mammalian cells. Somat Cell Mol Genet. 1998; 24:363-369.
Balciuniene, J., Nagelberg, D., Walsh, K.T., Camerota, D., Georlette, D., Biemar, F., et al. Efficient disruption of Zebrafish genes using a Gal4-containing gene trap. BMC Genomics. 2013; 14:619.
Romano, G., Marino, I.R., Pentimalli, F., Adamo, V., and Giordano, A. Insertional mutagenesis and development of malignancies induced by integrating gene delivery systems: implications for the design of safer gene-based interventions in patients. Drug News Perspect. 2009; 22:185-196.
Dupuy, A.J. Transposon-based screens for cancer gene discovery in mouse models. Semin Cancer Biol. 2010; 20:261-268.
Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G., and Jenkins, N.A. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature. 2005; 436:221-226.
Dupuy, A.J., Clark, K., Carlson, C.M., Fritz, S., Davidson, A.E., Markley, K.M., et al. Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci U S A. 2002; 99:4495-4499.
Dupuy, A.J., Fritz, S., and Largaespada, D.A. Transposition and gene disruption in the male germline of the mouse. Genesis. 2001; 30:82-88.
Clark, K.J., Carlson, D.F., Foster, L.K., Kong, B.W., Foster, D.N., and Fahrenkrug, S.C. Enzymatic engineering of the porcine genome with transposons and recombinases. BMC Biotechnol. 2007; 7:42.
Ivics, Z., and Izsvak, Z. Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies. Methods Mol Biol. 2004; 260:255-276.
Liu, H., Liu, L., Fletcher, B.S., and Visner, G.A. Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. FASEB J. 2006; 20:2384-2386.
Ohlfest, J.R., Lobitz, P.D., Perkinson, S.G., and Largaespada, D.A. Integration and long-term expression in xenografted human glioblastoma cells using a plasmid-based transposon system. Mol Ther. 2004; 10:260-268.
A second paper, by Larry Shook and Geoffrey Clark’s groups describe the production of ex vivo transformed porcine breast cancer line, driven by inactivation of BRCA1. In this paper normal porcine breast epithelial cells were immortalized by transfection with SV large T antigen (SV-LT) and upon inactivation of porcine BRCA1 in these immortalized cell lines, developed phenotype characteristic of transformed cells and exhibited cancer stem cell characteristics. The end point assay for transformation was growth in soft agar however the authors did not confirm malignancy in either SCID mice or SCID pigs.
Front Genet. 2015 Aug 25;6:269. doi: 10.3389/fgene.2015.00269. eCollection 2015.
A porcine model system of BRCA1 driven breast cancer.
BRCA1 is a breast and ovarian tumor suppressor. Hereditary mutations in BRCA1 result in a predisposition to breast cancer, and BRCA1expression is down-regulated in ~30% of sporadic cases. The function of BRCA1 remains poorly understood, but it appears to play an important role in DNA repair and the maintenance of genetic stability. Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene in vivo. However, the subtle nature of BRCA1 function and the well-known discrepancies between human and murine breast cancer biology and genetics may limit the utility of mouse systems in defining the function of BRCA1 in cancer and validating the development of novel therapeutics for breast cancer. In contrast to mice, pig biological systems, and cancer genetics appear to more closely resemble their human counterparts. To determine if BRCA1 inactivation in pig cells promotes their transformation and may serve as a model for the human disease, we developed an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line.
Figure 1. Immortalization of pig mammary epithelial cells. Primary pig breast epithelial cells were stably transfected with an SV40 LT expression construct and selected in puromycin. Surviving cells were serially passaged to confirm immortalization.
Figure 3. Loss of BRCA1 enhances pig mammary epithelial cell growth. (A) Serially passaging the pig mammary epithelial cells stably knocked down for BRCA1 resulted in an altered morphology compared to those cells stably expressing the LacZ miRNA. (B) 2 × 104 cells/well were plated in 6-well plates and cell growth was determined by counting the number of cells at the indicated times. Error bars show standard error, p < 0.05.
Figure 5. Loss of BRCA1 enhances the transformed phenotype of pig mammary epithelial cells. (A) The pig breast epithelial cells stably expressing BRCA1 miRNA were plated in soft agar and scored for growth 14 days later. Representative photomicrographs are shown in the top panel and data from three independent experiments quantitated in the bar graph in the lower panel. (B) 1 × 106 cells/well were plated in polyHEMA-coated 12-well plates and cell viability assessed 48 h later by trypan blue staining. Error bars show standard error, p < 0.05.
A third paper describes the development, in Gottingen minipigs, of a transgenic inducible model of intestinal cancer.
Mol Oncol. 2017 Nov;11(11):1616-1629. doi: 10.1002/1878-0261.12136. Epub 2017 Oct 10.
A genetically inducible porcine model of intestinal cancer.
Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT – which inhibits p53 – and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.
Other posts on this site related to Cancer Research Tools include
Many worldwide regulatory bodies are in agreement that proper choice of animal model is necessary for adequate extrapolation of toxicity and efficacy data from animal to human, considering the varied classes of therapeutics now being developed for oncology. The inability of screens, reliant on human xenografts grown in immunocompromised mice to evaluate host-immune and species-dependent effects, has made development of alternative animal-models a priority. This is evident in the fact that ninety percent of new anticancer drugs which showed anti-tumor efficacy in mouse preclinical models failed in human clinical studies. A recently developed “humanized” mouse model may assist in testing the metabolism of cancer drugs but still relies on older “immunosuppression” mouse models (http://stehlin.org/mouse-model-development/). This inadequacy of older, accepted models is clearly evident when evaluating safety and efficacy of adenoviral based gene therapies such as oncolytic conditionally-replicative adenovirus (CRAd). Although new-generation CRAds present with a relative safe profile[2, 3], adenoviral particles, especially the Ad5 based virus used for most CRAds, have the tendency to replicate in non-tumor tissue, such as liver and lung, resulting in tissue-specific toxicities[4-7]. The manifestation of these toxicities is only evident in species permissive for viral replication, such as the pig. Indeed, one of the first clinical trials with older adenovirus gene therapy, resulting in severe hepatic toxicity and fatality, may have been prevented if more appropriate preclinical screens were conducted. Thereafter, strict regulatory guidelines for adenoviral-based clinical trials have been issued, with particular emphasis on vector dosage, safety and toxicity[8]. Indeed, at Schering-Plough, a toxicology program was initiated to evaluate SCH 58500, and adenoviral gene therapy directed against p53, which involved use of non-immunogenic rats compared with testing in Yorkshire pigs made immunoreactive to the vector[9, 10]. In fact, data from the pig study revealed a faster clearance of virus as well as toxicities not seen in non-immunogenic, non-permissive hosts such as rat and mouse.
Therefore, development of a porcine model of cancer would permit both testing of both the efficacy and safety of these therapies in the same animal.
Development of large animal models of cancer
To date, large animal tumor models have been used for studying spontaneously formed tumors in dogs and cats [11](Vail, 2000, Cancer Invest), the most common being canine [12] and feline non-Hodgkin’s lymphoma [13]. The advantages of these companion models are the outbred nature of the animals, comparable size and kinetics to human tumors [14-18], and high incidence rates. Allografts of the outbred-canine transplanted venereal tumor have been used to test the ability to detect tumors using X-ray computed tomography and MRI with the ultimate goal of imaging-guided intervention. Researchers have recently utilized the spontaneously arising canine and feline soft tissue sarcomas to study effects of hyperthermia on chemotherapy pharmocokinetics, development of hypoxic cell markers, and cancer imaging techniques [15, 19-26]
Although it appears that, for a select number of tumor types, spontaneously arising tumors in large outbred animals can be useful to model the human disease, it is disappointing these spontaneous arising tumors are limited to discrete tumor types. However, due to recent advances in sequencing of several domestic animal genomes and the development of new cloning strategies, it is now very feasible to utilize other animal models more relevant to human disease, notably the miniature pig.
The Gottingen mini-pig
Large animals in medical research: Advantages of the minipig
Due to recent advances in sequencing of several domestic animal genomes [27, 28] and the development of new organism cloning technologies [29-31], it is now very feasible to utilize other species to model human disease, notably the pig. The development of porcine models of human disease has gained much interest lately. Advantages include the resemblance in anatomy, physiology, and genetic makeup with the human, as well as new methods to manipulate the pig genome [32, 33]. To date, porcine models of human metabolic syndrome [34] and diabetes [35], aortic aneurism [36], infectious disease resistance [32, 37], seizure [38], neurologic syndromes [33], and pancreatitis [39] have been developed. Recently, a genetically-engineered porcine model of cystic fibrosis was produced in collaboration with investigators at University of Iowa and Exemplar Genetics [40-42]. Additionally, Cho et al. successfully transplanted spontaneously transformed leukemic and lymphatic tumor cells in a major histocompatibility complex (MHC)-defined inbred miniature swine model [43], suggesting feasibility of an ex vivo strategy to develop a porcine tumor model. Porcine models have, also, been used to develop, test and refine surgical [44, 45] and laparoscopic techniques [46, 47], radio- and cryoablation protocols of tissues [48-52] and robotic surgery using the da Vinci Surgical SystemÒ [53, 54]. In addition, because of the size of porcine organs and their resemblance to the human (in genetics) the minipig is very useful and abundant of a source to isolate specific cell types for in vitro studies. Below is a figure showing the comparable size of human and porcine ovaries to the mouse and ability to purify porcine ovarian epithelial cells and their similarity to human and mouse ovarian epithelial cells.
Figure 1. The human and pig ovary have similar size and can yield a greater number of isolated cells than one can get from a mouse ovary.
Figure 2. Isolation and morphology of ovarian epithelial cells from three sources:
To date, there has been no allograft or xenograft model of cancer in pigs. The consensus amongst many surgeons suggests development of a minipig tumor model would be an invaluable tool for developing surgical skills.
A recent advancement in porcine tumor modeling was made by collaboration between researchers from the laboratories of Dr. Stefan Bossmann and Deryl Troyer at Kansas State and Iowa State, respectively[1]. The joint collaboration resulted in the development of the first severe combined immunodeficient pig line (SCID pig) which was shown to be able to accept human tumor xenografts. The line of immunodeficient pig was discovered when Yorkshire pigs were bred for increased feed efficiency and a line of pigs exhibited SCID-like symptoms including:
Decreased levels of circulating lymphocytes
Atrophied thymus and lymph nodes
The SCID phenotype in mice have been ascribed to defects in a DNA-dependent protein kinase gene which prevents variable-diversity-joining [V(D)J] gene region recombination[55]. There have been multiple genetic defects found in humans resulting in SCID, including defects in adenylate kinase2, Janus kinase 3, the IL2 receptor, and the IL-7 receptor[56]. The SCID phenotype in this pig line has a simple autosomal recessive inheritance pattern which, as described below in an interview with the authors, allows for the propagation of this porcine line.
An important feature of SCID models is the ability of these animals to act as a recipient of human tumorigenic cell lines. In fact, growth of cell lines in SCID mice is a common test for tumorigenicity. Therefore, to test if these pigs could act as recipients for human cancer cell lines, the authors inoculated the SCID Yorkshire pigs with 4 million A3755M human melanoma cells or PANC1 human pancreatic carcinoma cells subcutaneously in the left and right ears respectively of three pigs. Some features of the results include:
All injection sites showed evidence (either histologic or palpable) of tumor growth
Tumors showed characteristic histologic features of malignant neoplasm including
Bizarre and atypical mitotic figures
Anisocytosis (different cell sizes and shapes; feature of malignancy)
tumors stained with anti-human mitochondrial antibody (a marker of epithelial cancer cells) showed strong cytoplasmic staining of neoplastic cells
interestingly no necrotic regions in the tumor
Figure 3. Visual evidence of human tumor cells growing in SCID pig ear (day 20). B) Same picture as A) but circle outlines growth. From reference 1. Basel et al., used with permission from Mary Liebert.
It is interesting to note that these tumors only grew roughly 10 x 5.5 mm, which is genrally large enough to do preclinical studies but may be too expensive to be of use for xenograft studies. However it would be very feasible to conduct allograft studies in these SCID pigs.
Dr. Jack Dekkers, C.F. Curtiss Distinguished Professor and Section Leader of Animal Breeding and Genetics at Iowa State University, was kind to answer a few questions about the SCID pig model.
Question: You had mentioned this line was identified after breeding Yorkshire pigs for increased feed efficiency. Have you identified or hypothesize which altered pathway or molecular defect which results in a SCID phenotype? Is this SCID phenotype a result of a metabolic syndrome these pigs could have?
Dr. Dekkers: We indeed identified the SCID phenotype in a line of pigs that we had selected for increased feed efficiency. However, I don’t think this phenotype has anything to do with the selection we practiced; it was either already present in the founders of the line or it was a random mutation that occurred in the line, independent of the selection for feed efficiency. We have narrowed the mutation that causes the SCID in our pigs down to a chromosomal region and have a very strong candidate gene in that region that we are currently pursuing.
Question: In your opinion, is it possible to produce a highly inbred immunocompromised strain of pig such as a Gottingen minipig?
Dr.Dekkers: We are working on breeding the SCID mutation into mini pigs. But in the meantime, we have used bone marrow transfer to create a male that is homozygous SCID (it’s an autosomal recessive) and reproducing. This allows us to produce litters that are 50% SCID and 50% normal (carriers) by mating him to carrier females.
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UPDATED 3/14/2020
A recent Research Article and Research Article Summary in Science discusses, by the primary author of her study that describes the utility of the pig as an excellent surrogate model of the human brain and human brain function. The study, by Dr. Evelina Sjostdedt et al., was an integrative analysis of porcine, mouse, and human transcriptomic, genomic, and proteomic data from discrete anatomical regions of the brain. The global analysis suggested that there is similar regional organization and expression patterns among the three mammalian species. The authors found interspecies variability with respect for many neurotransmitter receptors.
However, for some regions of the brain, such as the cerebellum and hypothalamus, the human global expression profile is closer to that of the pig than of the mouse, suggesting that the pig might be considered a preferred animal model to study many brain processes.
In addition, interestingly, the authors found that many signature genes canonically thought to only be expressed in certain brain cells (astrocytes, microglia, oligodendrocytes) are expressed in higher levels in peripheral organs as well as immune cells.
to access the data used in this study, which includes high resolution images and metadata have also been made publicly available in the open-access Human Protein Atlas (HPA) Brain Atlas. at www.proteinatlas.org.
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