Enhancing Therapeutic Efficacy: Pioneering Large-Scale Drug Pipeline Development and Testing on PKU Patients' iPSC-Derived Hepatocytes
We are pleased to announce a new research project that we are funding lead by Michael Nafisinia. Sydney Medical School. University of Sydney.
PKU research summary -Amino Acid Metabolism Disorder group- Westmead Institute for Medical Research
The genotype of a patient is recognised as the primary determinant of the clinical features (mild, moderate or classic), as well as their responsiveness to treatment. Currently, study on the pathogenesis of amino acid metabolism disorders is limited, which is partly due to the lack of appropriate disease models, and those that can mimic the pathology of the disease in vitro and in vivo. In clinical settings, obtaining patient cells, like hepatocytes, neural cells are also difficult. The recent advent of induced somatic cell reprogramming with a combination of factors has allowed different disease-related cells to be obtained from induced pluripotent stem cells (iPSCs). The iPSC lines derived from patients’ tissues (blood, fibroblast, urine) can be efficiently directed differentiate into the cell type of interest. This provides an opportunity for detailed investigation of pathological processes in human tissues, which would not be generally accessible pre-mortem thus providing a disease model.
Since the proximate cause of most amino acid metabolism disorders is a deficiency of enzymes in the liver, patient liver cells have been chosen as the disease model to test for drugs. These samples will include patients from all age groups. Each sample will represent a different genotype causing the amino acid metabolism disorders. With the knowledge of the genetic information of a large cohort of patients we aim to building the very first cell biobank from patients with phenylketonuria (PKU) worldwide. Then with access to the patient cell biobank and a well-established drug-testing pipeline, we aim to achieve high-throughput screening of potential therapeutic agents.
Since 2018, the Amino Acid Metabolism Disorder Group, supported by the PKU Association of NSW, has taken on a unique approach. Departing from the conventional approach of conducting basic research and single drug testing for PKU, our focus has rapidly shifted towards the establishment of a patient cell biobank. This unique initiative involves utilising PKU patients with known genetic mutations.
Given the challenges associated with obtaining liver cells directly from patients—requiring invasive procedures under full anaesthesia, posing potential risks. Unlike many research groups worldwide, the amino acid metabolism disorder group, supported by the PKU Association of NSW has adopted an innovative strategy. Instead of relying on liver cells, we employ patient's skin fibroblasts and blood cells. By employing the advanced technique of iPSCs, we have the capability to generate liver cells which are the crucial cell type for PKU research.
Our previous achievements include the development of a robust screening methodology for cells treated with various drugs/compound or reagents, employing various techniques, such as:
Generation of genetically modified Crispr Cas 9 liver cell lines
Generation of iPSCs from blood cells and skin fibroblasts
Generation of liver cells from iPSCs
Establishment of an iPSCs biobank at the Westmead Institute for Medical Research, currently featuring six PKU cell lines and one health control sample
Quantification of mutated phenylalanine hydroxylase (PAH) enzyme transcript levels in liver cells utilising reverse-transcription PCR (RT-PCR)
Implementation of Immunofluorescent assays (IFA)
Employment of Immunoblotting to measure the phenylalanine protein abundance after and before treatment, reflecting the PAH enzyme activity.
Utilisation of Mass Spectrophotometry to determine phenylalanine hydroxylase activity in liver cells in converting amino acid phenylalanine to tyrosine.
Our strategic objectives for the next five years are as follows:
Expand the collection of iPSCs from PKU patients, focusing on the most common mutations globally. These lines will be incorporated into our biobank, providing a sustainable resource for ongoing research.
Establish a comprehensive and large-scale drug testing pipeline capable of efficiently screening hundreds of drugs within a condensed timeframe (months). This accelerated process contrasts with the prolonged timelines typically associated with traditional single drug testing over the course of several years
Establish collaborative partnerships with research centers and institutes, facilitating the sharing of our iPSCs cells with PKU research groups. This collaborative effort aims to advance gene therapy and drug development, promoting collective expertise in screening and refining potential treatments for PKU.
In pursuit of these objectives, our efforts aim to:
A. Expedite the discovery of personalised treatments for PKU patients carrying various mutations, with the ultimate goal of identifying a cure through a gene therapy approach.
B. Promote collaboration with other research groups, encouraging direct engagement with patients' PKU cells through our biobank, as opposed to using manipulated healthy cells.
These approaches, to the best of our knowledge, are highly distinctive to our group with the goal of addressing challenges in PKU research and establishing a gold standard for research in other genetic disease areas as a successful model.