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Richard A. Peterson II, DVM, PhD, Diplomate ACVP

AbbVie, Global Preclinical Safety, Investigative

Toxicology and Pathology

18 September 2017

Overview

Humanized mouse models (highly immunodeficient mouse strains) Mouse strains utilized

○ Humanized immune system

○ Humanized liver

○ Other

Infectious Disease and Husbandry Bacterial

Mycotic

Viral

Immune Mediated Disease GVHD

Other Musculoskeletal system changes

Pathology of Humanized Mice

In general pathologic changes in

humanized mice can be attributed to the

following:

Immune deficient state of mouse strains

(i.e., opportunistic infections, etc.)

Genetic background (i.e., gene

knockout/deficiency)

Humanization process (i.e., immune-

mediated disease/GVHD)

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Humanized Immune System

Mouse Models

Major mouse strain platforms for immune system humanization: NSG (NOD.Cg-PrkdcscidIL2rgtmlWjll/Sz)

NOG (NOD.Cg-PrkdcscidIL2rgtmlWjll)

NRG (NOD.129S7(B6)-Rag1tmlMomIL2rgtmlWjll/Sz)

BRG [C.129(Cg)Rag2tmlFwaIL2rgtmlSug/Jic]

NOD/SCID (NOD.CB17-Prkdcscid)

BLT (bone marrow-liver-thymus transplant in any of the above strains)

Next generation humanized mice: NSG mice

○ With murine MHC class I knockout

○ With human MHC class I and/or MHC class II transgenes

○ With human KitW-41

Allows more complete functional humanization and mitigation of graft-versus-host disease (GVHD)

Conditioning of the recipient mouse: irradiation, busulfan treatment

Humanized by intravenous or intrasplenic injection of human CD34+ hematopoietic stem cells (HSC)

Post-Humanization Immune

Tissue Morphology Injected human CD34+ HSC engraft the bone marrow of

recipient mice → human T-cells, B-cells, monocytes/

macrophages, NK cells, and dendritic cells which populate

the primary and secondary lymphoid tissues and periphery.

Normal cellular organization in secondary lymphoid tissues

(spleen and lymph nodes) is lacking. B-cells and T-cells are

present in these organs, but are intermingled (in sheets), not

in appropriate niches (e.g., PALS and lymphoid follicles).

Humanized Liver +/- Immune

System Mouse Models Humanized chimeric mouse models have been developed that may

be useful for the early characterization of human metabolism, assessments of safety for new drug candidates and preclinical models of human immunity.

Highly immunodeficient mouse strains allow reconstitution with human hepatocytes and/or immune cells..

Livers contain a chimeric mixture of native murine and reconstituted human hepatcocytes

Three common models:

uPA+/+ / SCID mouse transplanted with pediatric to adult human hepatocytes (PhoenixBio, University of Alberta)

Fah-/-/Rag2-/-/γC-/- mouse transplanted with pediatric to adult human hepatocytes (Hepatomouse™, Yecuris/Oregon Stem Cell Center, Oregon Health Sciences University)

Rag2-/-/γC-/- mouse (BalbC background) transplanted with human liver progenitor cells and CD34+ human hematopoeitic stem cells (University of North Carolina-Chapel Hill)

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Characteristics of the models High human hepatocyte replacement % (70-90+), but in large multilobular

to coalescing areas (bile connectivity/perfusion issues? No human immune system or adaptive mouse immune system)

uPA +/+ / SCID model transplanted with pediatric human hepatocytes (PhoenixBio, University of Alberta)

Fah-/-/Rag2-/-/γC-/- mouse transplanted with pediatric human hepatocytes (Hepatomouse™, Yecuris/Oregon Stem Cell Center, Oregon Health Sciences University)

Lower human hepatocyte replacement % (5-30%), but individualized human hepatocytes confined to smaller multifocal areas (more integrated structurally with a human immune system)

Rag2-/-/γC-/- mouse transplanted with human liver progenitor cells and CD34+ human hematopoeitic stem cells (University of North Carolina-Chapel Hill)

Infection with HCV:

uPA +/+ / SCID, Fah-/-/Rag2-/-/γC-/- , and Rag2-/-/γC-/- with human liver progenitor cells and CD34+ human hematopoeitic stem cells (i.e., supports HCV infection and fibrosis develops).

uPA+/+ SCID mouse, non-transplanted with neonatal human hepatocytes (control).

uPA+/+ SCID mouse, transplanted with neonatal human hepatocytes.

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uPA+/+ SCID humanized mouse

Liver, Anti-human Albumin IHC

uPA+/+ SCID Mouse transplanted with human hepatocytes, human region

CD10 IHC, interface

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MRP2 IHC, human area

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TEM, interface between mouse and human areas

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Fah-/-/Rag2-/-/γC-/- mouse

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Yecuris Mouse, FAH IHC. Human

hepatocytes are labeled for FAH

uPA+/+ SCID Fah-/- Rag2-/- γC-/-

Mutation Urokinase plasminogen activator over-

expression (in utero)

Fumaryl acetoacetate hydrolase

deficiency (inducible; NTBC

withdrawal)

Extensive Liver Humanization +

3-97% (average = 42%)

+

Up to 99%

Controllable Selection - +

Genotype Reversion + -

Age of Transplant

(mouse host)

Only young

(pre-weaning)

Any age

Breeding Efficiency Low High

Bleeding + -

Renal Damage (after repopulation) + -

Serial Transplantation - +

Etiologic agent susceptibility HBV, HCV, plasmodium HBV, HCV

Human liver disease correlates No fibrosis or HCC No fibrosis or HCC

Comparison of uPA and Fah Models

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Liver: Areas of mouse hepatocyte loss with

hemosiderin pigment-laden macrophages

and pale hepatocytes which are immunoreactive

for human albumin (red).

Rag2-/-/γC-/- mouse (BalbC background)

transplanted with human liver progenitor cells

and CD34+ human hematopoeitic stem cells

(University of North Carolina-Chapel Hill)

Animal No. 40, H&E. Activated caspase 3 IHC (insets)

Liver: Top Left: Small groups of

hemosiderin pigment-laden cells.

Macrophages vs. immature hepatocytes??

Bottom Right: Focal area of mouse

hepatocyte loss, inflammation with

lymphocytes, fibrosis, and occasional pale

irregular hepatocytes which are positive for

human albumin (red).

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Liver: Note the rare hepatocytes that express human

albumin (red), which have intracellular lipid droplets.

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Spleen: Top: Large pale cells within

spleen =human hepatocytes,

macrophages, or smooth muscle

cells? Bottom Left: note the lack of

human albumin immunoreactivity,

cells likely not human hepatocytes.

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Other Humanized Mouse Models

(examples)

AAD mice: transgenic for human HLA-

A2.

Alzheimer’s disease:

Transplanted neurons from AD patients

(Cahill and Huang, 2017)

Sjögren’s Disease Model (Young et al,

2015)

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Infectious Disease Concerns with

Humanized Mouse Models Highly immunodeficient mouse strains are extremely susceptible to infectious disease

caused by pathogenic and commensal bacteria: Corynebacterium bovis: “Coryneform hyperkeratosis”; Orthokeratotic

hyperkeratosis, epidermal hyperplasia, epidermal coryneform bacteria.

Pseudomonas aeruginosa (drinking water contamination, prevented by water

acidification/chlorination. Hepatic and splenic vasculitis, thrombosis, necrosis, hemorrhage)

Staphylococcus aureus/Coagulase-negative Staphylococcus spp. (periorbital abscessation, muzzle folliculitis/furunculosis, lacrimal and preputial gland

abscesses)

Pasteurella pneumotropica (commensal/opportunist in immunocompetent mice.

Immunosuppression or immunodeficient strains → abscessation of lymph nodes,

bronchopneumonia, metritis/accessory sex gland inflammation)

Corynebacterium kutscheri (hepatic and renal abscessation, suppurative arthritis)

Escherichia coli (hyperplastic typhlocolitis with mucosal erosions)

Citrobacter spp.

Enterobacter spp.

Proteus spp.

And opportunistic bacteria (ascending urinary tract, skin, abscesses, otitis media,

conjunctivitis, and oral infections; breeding/lactating females more susceptible):

Klebsiella oxytoca

Enterococcus spp.

Corynebacterium bovis, Nude mouse example.

Acanthosis, orthokeratotic

hyperkeratosis, surface

coryneform bacteria.

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Mouse, Staphylococcus spp., abcessation, head/cervical area

*

Gram Stain

Staphylococcus spp.

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Spleen, extramedullary

granulopoiesis Lung, pyogranuloma

Tail, pyogranuloma

Mouse, Staphylococcus spp., abcessation and myeloid hyperplasia/EMH

Infectious Disease: Mycotic agents

Mycotic agents

Pneumocystis murina (carinii)

○ A saprophytic protist fungus that causes lung infection in immunodeficient mice (SCID>Nude)

○ Asexual (trophozoites) and sexual (cysts) phases in life cycle

○ Usually asymptomatic in immunocompetent mice

○ Severe disease in SCID mice

Progressive dyspnea → death

Alveoli contain “foamy” proteinaceous material that contain the cystic form (3-5μm diameter), trophozoites attach to pneumocytes by filopodia

Interstitial alveolar mixed inflammation (variable), consolidation

Pneumocystis murina (carinii)

GMS

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Infectious Agents: Viral

Murine Norovirus (MNV): wasting, diarrhea, and death. Microscopically, hepatitis,peritonitis, and interstitial pneumonia seen in immunodeficient animals. The infection is persistent with fecal shedding for months following infection.

Graft-Versus-Host Disease

(GVHD) Occurs in NOD-SCID mice, BLT, and NSG mice

reconstituted with human CD34+ HSCs.

Human immune response against mouse tissues (MHCI-

based).

Lymphocytic infiltrate in several organs including skin,

conjunctiva, parotid salivary gland, colon, and lung

Other:

Musculoskeletal changes: Proliferative cartilaginous lesions

○ Recently described in humanized NOG mice (Gains et al 2017).

○ 2/9 humanized NOG mice, 26 weeks of age, treated with a proprietary immunomodulator had unilateral swelling of the tibiotarsal joint.

○ Chondroid metaplasia of the periosteum of the talus and calcaneal tendon with focal heterotopic ossification in a singlem animal.

○ No evidence of infectious agents.

Heterotopic ossification of the gastrocnemius tendon (MacLeod et al 2017) ○ Recently described in NOG mice

○ 2-3 months of age, unilateral or bilateral swelling of the tibiotarsal joint, 23/56 mice affected.

○ Cartilage and woven bone within the gastrocnemius tendon → hock swelling.

○ No evidence of infectious agents.

○ Uncertain cause, but strain is susceptible.

Described simultaneously. Likely reflect the same lesion in NOG mice, humanization seems to not be a cause, likely related to the genotype of the strain.

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