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Freezing characteristics of genetically modified lymphocytes for the treatment of MPS II
A Hubel1, T B Darr, J A Norman
1Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA. hubel001@maroon.tc.umn.edu
Abstract:
The freezing characteristics of genetically modified lymphocytes obtained from a donor with mucopolysaccharidosis type II (MPS II) were determined using cryomicroscopy and controlled rate freezing studies to determine postthaw viability. The cells from a donor with MPS II used in this investigation were cultured and transduced with a retroviral vector for the iduronate-2-sulfatase (IDS) enzyme for clinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics of the cells were determined after completion of the culture and transduction protocol. The water transport parameters, I(pg) and E(lp), for the cultured and transduced cells were determined to be 4.4 +/- 1.3 x 10(-14) m3/Ns and 173 +/- 25 kJ/mol, respectively. The IIF nucleation parameters, kappa and omega, were 5.5 x 10(10) K5 and 3.5 x 10(11) (l/m2 s), respectively. The postthaw viability of the genetically modified cells was less than the viability of the freshly isolated cells from the same donor. The postthaw viability of the cultured and transduced cells from a donor with MPS II was also less than that observed with cells from a normal donor that were frozen and thawed under the same conditions. These studies are essential in understanding the biophysical changes resulting from the ex vivo culture of cells and the manner in which these changes influence the ability of the cells to be cryopreserved.
Insights
Genetically modified lymphocytes for mucopolysaccharidosis type II (MPS II) gene therapy showed reduced post-thaw viability. Cryopreservation challenges stem from biophysical changes during ex vivo cell culture and transduction.
Area of Science:
- Cellular cryobiology
- Biophysics of cell freezing
- Gene therapy for rare diseases
Background:
- Mucopolysaccharidosis type II (MPS II) is a genetic disorder requiring innovative treatments.
- Human gene therapy aims to restore enzyme function using genetically modified cells.
- Ex vivo cell manipulation can alter cellular properties, impacting cryopreservation.
Purpose of the Study:
- To assess the cryopreservation characteristics of genetically modified lymphocytes from an MPS II donor.
- To determine the influence of ex vivo culture and transduction on cell water transport and intracellular ice formation (IIF).
- To evaluate the post-thaw viability of these modified cells for potential gene therapy applications.
Main Methods:
- Controlled rate freezing and cryomicroscopy were employed.
- Water transport parameters (I(pg), E(lp)) and IIF nucleation parameters (kappa, omega) were quantified.
- Post-thaw viability was compared between modified, fresh, and normal donor cells.
Main Results:
- Cultured and transduced MPS II lymphocytes exhibited altered water transport (I(pg) = 4.4 ± 1.3 x 10⁻¹⁴ m³/Ns, E(lp) = 173 ± 25 kJ/mol) and IIF nucleation (kappa = 5.5 x 10¹⁰ K⁵, omega = 3.5 x 10¹¹ (l/m²s)).
- Post-thaw viability of genetically modified cells was lower than fresh cells from the same donor.
- Viability was also reduced compared to cryopreserved cells from a normal donor.
Conclusions:
- Ex vivo cell culture and genetic modification protocols significantly impact lymphocyte cryopreservation.
- Biophysical changes induced by these protocols compromise cell survival post-thaw.
- Understanding these changes is crucial for optimizing cell-based gene therapies for MPS II and similar conditions.