RESUMO
CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.
Assuntos
Hemoglobina Fetal/genética , Filtração/métodos , Edição de Genes/métodos , Transplante de Células-Tronco Hematopoéticas , Ribonucleoproteínas/genética , Animais , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Eletroporação/métodos , Feminino , Hemoglobina Fetal/metabolismo , Filtração/economia , Filtração/instrumentação , Terapia Genética/economia , Terapia Genética/instrumentação , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Humanos , Membranas Artificiais , Camundongos , Modelos Animais , RNA Guia de Cinetoplastídeos/genética , Transplante AutólogoRESUMO
A hindered urea bond (HUB), recently reported as a new type of dynamic chemical bond, can be facilely constructed by mixing an isocyanate and a hindered amine. Here, we report the use of the HUB in the design of degradable hydrogel materials for applications of stem cell encapsulation and delivery. Polyethyleneglycol (PEG) diamine was end-capped with a HUB and an allyl group in a one-pot synthesis. The resulting polymer was cross-linked to form a hydrogel under UV with the addition of a 4-arm PEG thiol and a photoinitiator. The degradation properties of the hydrogels were confirmed with NMR, GPC, weight loss, and protein release studies. We found that the degradation kinetics is dependent on the size of the N-substituents, and the one with the tert-butyl group shows complete degradation within 2 days. The new hydrogel materials were also demonstrated to be biocompatible with hMSCs, and the cell release kinetics can be facilely tuned over 5 days.
Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Ureia/química , Aminas/química , Materiais Biocompatíveis/uso terapêutico , Humanos , Hidrogéis/uso terapêutico , Hidrólise , Isocianatos/química , Cinética , Polietilenoglicóis/química , Polímeros/química , Proteínas/química , Compostos de Sulfidrila/químicaRESUMO
We report the design and development of redox-responsive chain-shattering polymeric therapeutics (CSPTs). CSPTs were synthesized by condensation polymerization and further modified with poly(ethylene glycol) (PEG) via "Click" reaction. Size-controlled CSPT nanoparticles (NPs) were formed through nanoprecipitation with high drug loading (up to 18%); the particle size increased in a concentration dependent manner. Drug release from particles was well controlled over 48 h upon redox triggering. The anticancer efficacy of the CSPT NPs was validated both in vitro and in vivo.