Plenary Session

Presenter 1

Introduction: Classical galactosemia (CG) is a hereditary disorder of the galactose metabolism caused by a severe deficiency of galactose-1-phosphate uridylyltransferase (GALT) enzymatic activity. The current standard of care, a galactose-restricted diet, fails to prevent chronic impairments affecting the brain and female gonads. Messenger RNA (mRNA) has emerged as a novel therapeutic approach especially interesting for monogenic disorders, such as inborn errors of metabolism. We have investigated the potential of GALT mRNA therapy in a zebrafish model of classical galactosemia. 

Methods: We injected galt knockout or wildtype zebrafish embryos at the one-cell stage with either naked or lipid nanoparticle (LNP)-packaged human GALT (hGALT) mRNA to restore GALT enzyme activity. A fraction of the embryos was exposed to galactose from 24 hours post fertilization. GALT enzyme activity, galactose-1-phosphate levels and toxicity were assessed at five days post fertilization. In addition, we injected embryos with green fluorescent protein (GFP) mRNA to examine differences in the distribution of naked vs. LNP-packaged mRNA. 

Results: Single injections with naked or LNP-packaged hGALT mRNA generated a dose-dependent increase in functional GALT enzyme. hGALT mRNA injections rescued galactose-1-phosphate levels in embryos exposed to galactose. LNP-packaged mRNA injections resulted in a higher GFP expression than naked mRNA injections and gave rise to a broad distribution of GFP signal. The mRNA injections did not have a toxic effect on morphology, survival or growth. Conclusion: Injections with human GALT mRNA resulted in a broad distribution of functional GALT enzyme in galt knockout zebrafish embryos. These promising results justify further research. Our next step will be to target the mRNA to one of the organs of damage in CG – i.e. the female gonads – and to investigate the window of therapeutic opportunity in galt knockout zebrafish. 

Presenter 2

Patients suffering from critical limb-threatening ischemia (CLTI) have a risk of undergoing a major amputation within 1 year of 30% with a substantial risk of undergoing re-amputation since wound healing is often impaired. Quantitative assessment of regional tissue viability following amputation surgery could identify patients at risk for impaired wound healing. To quantify regional tissue perfusion, near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) seems promising.

This pilot study included adult patients undergoing amputation surgery due to CLTI or diabetes mellitus. ICG-NIR fluorescence imaging was performed within 5 days after surgery using the Quest Spectrum Platform®. Following intravenous administration of ICG, ICG-NIR fluorescence was recorded for 10 minutes. These videos were analyzed and if a fluorescence deficit was observed, the area was marked as “low fluorescence”. All other regions were marked as “normal fluorescence”.

Successful ICG-NIR fluorescence was performed in 10 patients undergoing a total of 15 amputations. No fluorescence deficits were observed in 11 out of 15 amputation wounds. In 10 out of 11, no wound healing problems occurred during follow-up. Regions with “low fluorescence” were observed in 4 amputation wounds and impaired wound healing corresponding to the area of these regions was observed in all, requiring re-amputation in 3 out of 4. Time-related parameters from video analysis demonstrated a significantly longer time to max intensity (113 seconds vs. 32 seconds, p=0.003) and a significantly lesser decline in outflow after five minutes (80.3% vs. 57.0%, p=0.003) in regions marked as low fluorescence.

In 4 out of 5 amputations, wound healing problems occurred due to non-viability of the surrounding skin. ICG-NIR fluorescence was able to predict postoperative complications in all cases. However, quantitative assessment using ICG-NIR fluorescence remains challenging due to influences of external factors. Prospective studies on inter-operative use of ICG-NIR are required to provide surgeons with reliable cut-off values. The potential prediction of wound healing using ICG-NIR fluorescence will have an enormous impact on patient mortality as well as the burden of amputation surgery on health care.