Endovascular transplantation of mRNA-enhanced mesenchymal stromal cells results in superior therapeutic protein expression in swine heart.

Heart failure has a poor prognosis and no curative treatment exists. Clinical trials are investigating gene- and cell-based therapies to improve cardiac function. The safe and efficient delivery of these therapies to solid organs is challenging. Herein, we demonstrate the feasibility of using an endovascular intramyocardial delivery approach to safely administer mRNA drug products and perform cell transplantation procedures in swine. Using a trans-vessel wall (TW) device, we delivered chemically modified mRNAs (modRNA) and mRNA-enhanced mesenchymal stromal cells expressing vascular endothelial growth factor A (VEGF-A) directly to the heart. We monitored and mapped the cellular distribution, protein expression, and safety tolerability of such an approach. The delivery of modRNA-enhanced cells via the TW device with different flow rates and cell concentrations marginally affect cell viability and protein expression in situ. Implanted cells were found within the myocardium for at least 3 days following administration, without the use of immunomodulation and minimal impact on tissue integrity. Finally, we could increase the protein expression of VEGF-A over 500-fold in the heart using a cell-mediated modRNA delivery system compared with modRNA delivered in saline solution. Ultimately, this method paves the way for future research to pioneer new treatments for cardiac disease.

Phenotyping of Macrophages After Radiolabeling and Safety of Intra-arterial Transplantation Assessed by SPECT/CT and MRI.

Cell therapy is an integral modality of regenerative medicine. Macrophages are known for their sensitivity to activation stimuli and capability to recruit other immune cells to the sites of injury and healing. In addition, the route of administration can impact engraftment and efficacy of cell therapy, and modern neuro-interventional techniques provide the possibility for selective intra-arterial (IA) delivery to the central nervous system (CNS) with very low risk. The effects of radiolabelling and catheter transport on differentially activated macrophages were evaluated. Furthermore, the safety of selective IA administration of these macrophages to the rabbit brain was assessed by single-photon emission computed tomography/computed tomography (SPECT/CT) and ultra-high-field (9.4 T) magnetic resonance imaging (MRI). Cells were successfully labeled with (111In)In-(oxinate)3 and passed through a microcatheter with preserved phenotype. No cells were retained in the healthy rabbit brain after IA administration, and no adverse events could be observed either 1 h (n = 6) or 24 h (n = 2) after cell administration. The procedure affected both lipopolysaccharide/gamma interferon (LPS/IFNγ) activated cells and interleukin 4 (IL4), interleukin 10 (IL10)/transforming growth factor beta 1 (TGFß1) activated cells to some degree. The LPS/IFNγ activated cells had a significant increase in their phagocytotic function. Overall, the major impact on the cell phenotypes was due to the radiolabeling and not passage through the catheter. Unstimulated cells were substantially affected by both radiolabeling and catheter administration and are hence not suited for this procedure, while both activated macrophages retained their initial phenotypes. In conclusion, activated macrophages are suitable candidates for targeted IA administration without adverse effects on normal, healthy brain parenchyma.

Comparative in vivo biodistribution of cells labelled with [89Zr]Zr-(oxinate)4 or [89Zr]Zr-DFO-NCS using PET.

BACKGROUND: In vivo monitoring of cell biodistribution using positron emission tomography (PET) provides a quantitative non-invasive method to further optimize cell therapies and related new developments in the field. Our group has earlier optimized and evaluated the in vitro properties of two radiotracers,[89Zr]Zr-(oxinate)4 and [89Zr]Zr-DFO-NCS, for the radiolabelling of different cell types. Here, we performed a microPET study to assess the in vivo biodistribution of cells in rats using these two radiotracers. Human decidual stromal cells (hDSC) and rat macrophages (rMac) were radiolabelled with [89Zr]Zr-(oxinate)4 or [89Zr]Zr-DFO-NCS. Rats were intravenously injected with radiolabelled cells, and the in vivo biodistribution was monitored with microPET/CT imaging for up to day 7. Organ uptake was evaluated and presented as a percentage of injected activity per gram tissue (%IA/g) and total absorbed organ doses (mSv/MBq). RESULTS: The biodistribution in vivo showed an immediate uptake in the lungs. Thereafter, [89Zr]Zr-(oxinate)4 labelled cells migrated to the liver, while the signal from [89Zr]Zr-DFO-NCS labelled cells lingered in the lungs. The differences in the in vivo behaviour for the same cell type appeared related to the radiotracer labelling. After 24 h, [89Zr]Zr-(oxinate)4 labelled cells had over 70% higher liver uptake for both hDSC and rMac compared to [89Zr]Zr-DFO-NCS labelled cells, whereas [89Zr]Zr-DFO-NCS labelled cells showed over 60% higher uptake in the lungs compared to [89Zr]Zr-(oxinate)4 labelled cells. This difference in both lung and liver uptake continued until day 7. Dosimetry calculations showed a higher effective dose (mSv/MBq) for [89Zr]Zr-DFO-NCS compared to [89Zr]Zr-(oxinate)4, for both cell types. Although the bone uptake was higher for [89Zr]Zr-(oxinate)4 labelled cells, the prolonged uptake in the lungs contributed to a significant crossfire to bone marrow resulting in a higher bone dose. CONCLUSION: The [89Zr]Zr-DFO-NCS labelled cells suggest a prolonged accumulation in the lungs, while [89Zr]Zr-(oxinate)4 suggests quicker clearance of the lungs followed by accumulation in the liver. Accumulation of radiolabelled cells in the liver corresponds to other cell-tracking methods. Further studies are required to determine the actual location of the [89Zr]Zr-DFO-NCS labelled cell.

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications.

BACKGROUND: There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. PROCEDURES: Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. RESULTS: The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1-8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells' proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. CONCLUSIONS: Our study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.