Requirements rev 5.pdf

PCA-NHGRI-0616 Statement of Work (SOW)

Study: Histopathology and antibody screening of Hutchinson Gilford progeria syndrome mouse model DNA editing therapeutic preclinical trial samples.

Background:

We are conducting a preclinical trial, using our humanized mouse model of Hutchinson Gilford Progeria Syndrome (HGPS), testing the efficacy of therapeutic adenine DNA base editing, administered using Adeno-Associated Virus 9 (AAV9), for correcting the mutation that causes HGPS. This preclinical research is being used to support the application for the FDA Investigational New Drug (IND), SamPro-2, to treat HGPS patients in a Phase 1/2 clinical trial. The FDA proposal requires demonstration of efficacy of the DNA editing and the demonstration of non-toxicity. The classical pathology of HGPS in humans and in the HGPS mouse model is the loss of vascular smooth muscle cells in the large arteries. The required demonstration of non-toxicity is the detection of AAV9 antibodies in the mouse serum six months after treatment. To satisfy the FDA requirements the pathology analysis and the toxicology analysis need to be conducted with Good Laboratory

Practice (GLP) compliance and records. The aim of this procurement is to perform mouse tissue histology processing and histopathology staining on the tissues of 72 mice, and AAV9 antibody screening of 60 mouse serum samples incorporating GLP-compliant documentation.

Purpose and Objectives:

The requirements for this procurement are providing critical pathology and assay analysis to support the SamPro-2 AAV9 gene editor program for Hutchinson-Gilford Progeria Syndrome (HGPS). The plan is structured to complement the work being conducted at the NIH and align with FDA recommendations to PRF for IND-enabling preclinical success.

This study will generate up to 18 fixed tissue samples from each of 60 mice to process for histopathology slides. Hematoxylin and eosin pathology staining will be performed on aorta, heart, liver, kidney, brain and lung. Movat's pentachrome staining will be performed on the aorta for vascular smooth muscle content and pathology analysis. Picrosirius red and alizarin red staining will be performed on the heart to examine for pathology. Unstained tissue slides of all tissues will be provided for immunofluorescence histopathology analysis by the Molecular Genetics section, NHGRI.

AAV9 antibody screening will be performed on each of 60 mouse serum samples using a proprietary assay qualified and approved for the detection of AAV9 antibodies by the FDA. The results of these histopathology examinations and antibody tests will be documented in a final report using GLP-compliant guidelines. All histopathology slides and tissue samples will be returned to the Molecular Genetics section, NHGRI, at the completion of the project.

All studies will be performed in the HGPS mouse model.

All drug product administration will be performed via retroorbital injection, at NHGRI

All aspects of the study will be performed as equivalent to GLP standards and procedures as possible.

Period of Performance:

Base Year: July 30, 2026 to December 31, 2026

Contractor’s Requirement and Deliverables:

Below we have outlined the proposed segments of the protocol that are required, with enough detail to derive a scope of work.

All tissue and blood samples remaining after the analyses below will be stored under appropriate conditions until returned to origin.

Section I. Efficacy and Toxicity Study (PRF-A-ETOX) – 6-month sub-study arm

A. NIH has injected and performed in-life on the mice.

B. NIH will necropsy the animals

C. NIH will provide organs/tissues after necropsy is performed at NIH and organs/tissues are placed into 2% paraformaldehyde.

1. The primary task is to perform ‘GLP-compliant’ pathology studies with full GLP-compliant reports.

2. Blinded slides will be sent back to the NIH, for NIH to then perform immunofluorescence histochemistry (IHC) evaluations of SamPro-2 efficacy.

• Total Animals: 60

• Groups: 5 dose groups, 12 animals per dose

• Sex Distribution: 12 animals per dose group (6 Males, 6 Females).

• Timing: Necropsies will occur August 14-28, 2026 and organs shipped thereafter.

• Total Number of Slides: 141x60=8460

Study Arm Group #s Test Agent Doses(vg/kg) Total Animals

6-Month Arm 6–10 Vehicle &

SamPro-2

5e12 to 1e14 60

Table 1 Paraffin embedding, microtomy and staining

Organ type # of mice

Slide prep Orientation # of Slides and Stains per organ in order of slice and stain

# of slide s/ mou se

# of section s per slide

Ascending

Aorta**

60 Section ascending aorta

1.5mm, 5 sections from aortic root, place aortic root offset the four remaining sections in proximal root to distal order transverse H&E=1

Movat’s Pentachrome=

Unstained=5 H&E=1

Movat’s Pentachrome=

9 5

Descending Aorta**

60 Section ascending aorta 1.5mm, 5 sections from aortic root, place aortic root offset the four remaining sections in proximal root to distal order transverse H&E=1 Movat’s Pentachrome=

Unstained=5

H&E=1 Movat’s Pentachrome=

9 5

Heart** 60 Section transverse, apex 1mm, 1/3 above apex, 2/3 above apex transverse, left ventricle focus

H&E=1 Picrosirius

Red=1 Alizarin Red=1

Unstained=5 H&E=1

Picrosirius Red=1 Alizarin

Red=1

11 3

Liver** 60 Standard pathology section, 1-2mm transverse H&E=1 Unstained=5

H&E=1

7 2

Left Kidney 60 Standard pathology section, 1-2mm longitudina l

H&E=1 Unstained=5

H&E=1

Hemisected Brain cortex***, 60 Standard pathology section, 1-2mm transverse H&E=1 Unstained=5

Hemisected Brain

60 Standard pathology section, 1-2mm transverse H&E=1 Unstained=5

7 2 cerebellum*

H&E=1

Hemisected

Brain brainstem**

60 Standard pathology section, 1-2mm transverse H&E=1

Unstained=5 H&E=1

7 2

Left Lung 60 Standard pathology section, 1-2mm transverse H&E=1

Unstained=5

Spleen** 60 Standard pathology section, 1-2mm transverse H&E=1

Unstained=5 H&E=1

7 2

Skin 60 Standard pathology section, 1-2mm transverse H&E=1

Unstained=5 H&E=1

7 2

Ovary (left;

females)

30 Standard pathology section, 1-2mm transverse H&E=1 Unstained=5

H&E=1

7 2

Testis (left;

male)

30 Standard pathology section, 1-2mm transverse H&E=1 Unstained=5

H&E=1

7 2

Cervical Dorsal root ganglia*

3 M/F/ cohort Total not applicable

(NA)

Collected and archived

0 2

Thoracic Dorsal root ganglia*

3 M/F/ cohort

Total

N/A Collected and archived

Lumbar

Dorsal root ganglia *

3 M/F/ cohort Total

N/A Collected and archived

Mandibular

Lymph nodes

3 M/F/ cohort Total

N/A Collected and archived

Femur, Left 60 Standard pathology section, 1-2mm

Longitudin al

H&E=1 Unstained=5

H&E=1

7 2

Quadricep, Left

60 Standard pathology section, 1-2mm transverse H&E=1 Unstained=5 H&E=1

7 2

Brown adipose

60 Standard pathology section, 1-2mm transverse H&E=1

Unstained=5

7 2

White adipose

60 Standard pathology section, 1-2mm transverse H&E=1 Unstained=5 H&E=1

7 2

Small Intestine

60 Standard pathology section, 1-2mm

Longitudin al

H&E=1 Unstained=5 H&E=1

7 2

Large intestine

60 Standard pathology section, 1-2mm

Longitudin al

H&E=1

Unstained=5 H&E=1

7 2

For aorta histology (performed at CRL), tissues will be fixed in 2% paraformaldehyde for 24 hours prior to dehydration with graded alcohols and embedded in paraffin. Cross-sections (4-μm thick) will be cut and mounted on charged slides and visualized by hematoxylin and eosin (H&E) staining.

Additional staining will be performed with Movat’s pentachrome (CRL).

*Collected and archived for future retrospective analysis

** these organs dissected at NIH into fixed portion sent to CRL and frozen portion kept at NIH

***- entire brain fixed and sent to CRL – CRL to divide into the 3 regions