Development of the UValidate platform for the profiling of topically applied chemical agents.

Project Summary
Skin is the first line of defense against the outside environment. When the skin barrier is breached by UV
irradiation (UVR) or toxic chemicals, living cell layers including epidermal keratinocytes, melanocytes and dermal
fibroblasts are subjected to DNA damage. If unrepaired, this damage can lead to photoaging and cutaneous
carcinogenesis. In fact, skin cancer is by far the most common malignancy, accounting for more cases of cancer
in the US and other countries than all other organs combined. Sunscreens have been used extensively with
some success, but newer chemical filters suffer from serious shortcomings that make currently available
products undesirable, and can be a risk to human health. Current strategies that examine the effectiveness and
safety of organic UV filters need to be reassessed. These new strategies should test the biology of UVR in the
presence of sunscreens. Further, they need to be high-throughput (HTP) in order to examine combinations of
different chemical blockers that are present in most formulations, work in cultured cells for ease of screening,
and be animal-friendly. In Phase I we showed proof-of-principle for a novel UVR HTP screening platform called
“UValidate, employing mixed populations of isogenic keratinocytes and melanocytes derived from a single donor,
exposed to two-compound combinations of sunscreen active ingredients in the presence and absence of short
wave (UVB; 285-320), and long wave solar UVR (UVA1; 340-400 nm). These screens measure reactive oxygen
species (ROS), DNA damage (CPDs), and micronuclei and show a complex interplay between solar UV
wavelength, sunscreen blockers, and cell types. This highlights the pressing need for our platform, compared
to currently-utilized assays that measure only the amount of UV that is blocked by spectrometry. In the proposed
Phase II, we plan to test known chemical UV blockers for the cosmetic and skin-care markets as a contracting
service, using a panel of donor-derived isogenic keratinocytes, melanocytes and fibroblast. At the completion of
Phase II, if funded, UValidate will be offered as a service particularly suited for companies at the early stages of
drug discovery, regulatory bodies and established pharma seeking to optimize formulation. Advances in this
proposal include fluoro-tagging isogenic patient cell lines, establishing 2D and 3D cell cultures to determine
individual responses to solar UV irradiation, utilization of reagents and assays to rapidly determine the types of
DNA lesions and their repair, and design of AI software to more accurately determine damage. Control cells,
generated using CRISPR technology, will reproduce DNA repair-compromised skin cells and 3D skin equivalents
will also be included to ensure experimental rigor and reproducibility. The present crisis due to the lack of non-
toxic sunscreens has led to class action lawsuits, banning of sunscreens in growing numbers worldwide, and
recall of formulations from the market. Therefore, this technology is timely and needed with great expediency to
address the urgent need for the discovery and testing of safe and effective UV blockers to prevent the most
common, and often debilitating and deadly cancers that we face.Development of the UValidate platform for the chemical profiling of topically applied chemical agents.
NARRATIVE
Over-the-counter (OTC) active ingredients that block UVA and UVB are required in all sunscreens. Recently,
the safety of these compounds has come under scrutiny both from a human health and ecological perspective.
In Phase I, we successfully developed the UValidate platform, to address the immediate need to develop a
better topical agent testing platform, particularly for UVA-blocking ingredients. UValidate is novel in that it can
test the genotoxicity of sunscreens in conjunction with UVR treatment. In Phase II, we will develop UValidate
from proof-of-principle, to a stable, integrated, HTP service platform able to measure biological endpoint
directly relevant to sunscreen efficiency using cutting-edge donor-derived cellular models and complex
combinational treatment regimes coupled with UVR exposure.