| 1. |
- Lundsten, Sara, et al.
(författare)
-
Tumor-Targeted Delivery of the p53-Activating Peptide VIP116 with PEG-Stabilized Lipodisks
- 2020
-
Ingår i: Nanomaterials. - : MDPI AG. - 2079-4991. ; 10:4
-
Tidskriftsartikel (refereegranskat)abstract
- Stapled peptides targeting the interaction between p53 and its negative regulators MDM2 and MDM4 have exhibited great potential as anti-cancer drugs, albeit with room for improvement in formulation and tumor specificity. Lipid bilayer disks (lipodisks) have emerged as promising drug nanocarriers and can by attachment of targeting moieties be directed selectively towards tumor cells. Tumor-targeted delivery of stapled peptides by use of lipodisks may therefore increase the uptake in the tumors and limit toxicity in healthy tissue. Here, we utilized epidermal growth factor receptor (EGFR)-targeted lipodisks to deliver p53-activating stapled peptide VIP116 to EGFR-expressing tumor cells. We demonstrate that VIP116 can be stably formulated in lipodisks (maximum peptide/lipid molar ratio 0.11). In vitro cell studies verify specific binding of EGF-decorated lipodisks to tumor cells and confirm that targeted delivery of VIP116 significantly decreases tumor cell viability.
|
|
| 2. |
- Molisak, Agnieszka, et al.
(författare)
-
CRISPR/Cas9 as a tool to disrupt wild-type and A53T SNCA in sporadic and familial Parkinson’s disease
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Parkinson’s Disease (PD) is characterized by pathological accumulation of α-synuclein (αSyn) as Lewy bodies and Lewy neurites in the brain. Current treatment strategies can only alleviate the symptoms but do not interfere with the disease progression. With the discovery of the CRISPR/Cas9 gene editing tool, it has become possible to target the generation of pathological protein aggregates at the DNA level. Disrupting the αSyn gene (SNCA) could prevent the formation of Lewy-related pathologies. Here, we have designed two CRISPR/Cas9-based approaches by using guide RNAs (gRNAs) that are targeting either wild-type (WT) SNCA (pan-SNCA) or mutant A53T SNCA that causes early-onset familial PD. We could demonstrate that plasmid vector-mediated transfection of the pan-SNCA gRNA led to robust allelic disruption in HEK293T cells and human fibroblasts and that the editing efficiency was further increased with the use of a lentiviral transduction system. In addition, the SNCA A53T gRNA was specific towards the mutation site, but resulted in low and inconsistent targeting efficiencies in human patient fibroblasts carrying the SNCA A53T mutation. Our results indicate that SNCA can be targeted by CRISPR/Cas9, although the system efficiency varies across different cell types. In the future, systemically administered gene-editing treatments based on CRISPR/Cas9 could provide a valid therapeutic approach for PD patients.
|
|
| 3. |
|
|
| 4. |
- Sarén, Tina, 1991-
(författare)
-
CAR T cells for Immunotherapy of Cancer
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In recent years, immunotherapy has revolutionized cancer treatment by prolonging survival and even curing patients lacking other available therapies. Besides immune checkpoint inhibitors, one of the major advances is attributed to the success of chimeric antigen receptor (CAR)-T cell therapy in treating patients with B-cell malignancies. Although many patients respond, some are resistant to treatment and others will relapse after an initial response.CAR-T cell efficacy can be enhanced by adding a transgene cassette encoding an immune-stimulatory molecule that induce bystander immunity and a broader anti-cancer response. This is particularly important to improve efficacy in solid tumors where the “CAR-target-antigen” is often heterogeneously expressed. The neutrophil-activating protein (NAP), a virulence factor of Helicobacter Pylori, is an efficient immune stimulator. When secreted from CAR-T cells NAP can induce bystander immunity with killing of “CAR-target-negative” tumor cells.In my thesis, I aim at investigating resistance mechanisms to CAR-T therapy and developing new improved CAR-T therapies.In paper I, clinical response in patients with B-cell lymphoma was evaluated after treatment with two doses of third-generation CD19CAR-T cells. Response was observed in 9 out of 24 patients (37.5%) irrespectively of receiving one or two doses. We also analyzed whether the profile of the individual CAR-T infusion products could predict response. CAR-Ts from responders had high cytotoxic and low dysfunctional profile while CAR-Ts from non-responders had high dysfunction profile. Extended culture time during manufacturing was associated with dysfunction. As CAR-T products of non-responders were on average cultured longer, this may explain their dysfunctionality. In paper II, we developed CD20-targeting CAR-T cells (CAR20-T), which efficiently killed CD20-expressing human lymphoma cell lines and patient-derived lymphoma cells in vitro. By arming CAR20-T cells with NAP we could delay tumor growth in lymphoma-bearing mice compared to conventional, unarmed CAR-T cells.In paper III, we engineered five IL13Rα2-targeting CAR-T cell constructs for glioblastoma therapy. The candidates only differed in the complementary determining regions (CDRs) of the single-chain variable fragment portion of the CAR. We found that CDR-mediated CAR clustering could lead to antigen-independent tonic signaling and subsequent CAR-T cell dysfunction. We also identified one candidate which did not display any significant tonic signaling and that possessed therapeutic effect on mice with orthotopic glioblastoma. IL13Rα2 is heterogeneously expressed in glioblastoma tumors so in paper IV, we armed the best CAR-T cell construct with NAP and found that NAP-armed IL13Rα2 CAR-T cells could prolong survival of glioblastoma-bearing mice.
|
|
| 5. |
|
|
| 6. |
- Sarén, Tina, et al.
(författare)
-
Complementarity-determining region clustering may cause CAR-T cell dysfunction
- 2023
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1, s. 4732-
-
Tidskriftsartikel (refereegranskat)abstract
- Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as cancer treatment, however, designing an optimal CAR remains challenging. A single-chain variable fragment (scFv) is generally used as CAR targeting moiety, wherein the complementarity-determining regions (CDRs) define its specificity. We report here that the CDR loops can cause CAR clustering, leading to antigen-independent tonic signalling and subsequent CAR-T cell dysfunction. We show via CARs incorporating scFvs with identical framework and varying CDR sequences that CARs may cluster on the T cell surface, which leads to antigen-independent CAR-T cell activation, characterized by increased cell size and interferon (IFN)-γ secretion. This results in CAR-T cell exhaustion, activation-induced cell death and reduced responsiveness to target-antigen-expressing tumour cells. CDR mutagenesis confirms that the CAR-clustering is mediated by CDR-loops. In summary, antigen-independent tonic signalling can be induced by CDR-mediated CAR clustering, which could not be predicted from the scFv sequences, but could be tested for by evaluating the activity of unstimulated CAR-T cells.
|
|
| 7. |
|
|
| 8. |
- Sarén, Tina, et al.
(författare)
-
Insertion of the Type-I IFN Decoy Receptor B18R in a miRNA-Tagged Semliki Forest Virus Improves Oncolytic Capacity but Results in Neurotoxicity
- 2017
-
Ingår i: MOLECULAR THERAPY-ONCOLYTICS. - : CELL PRESS. - 2372-7705. ; 7, s. 67-75
-
Tidskriftsartikel (refereegranskat)abstract
- Oncolytic Semliki Forest virus (SFV) has been suggested as a potential candidate for the treatment of glioblastoma and neuroblastoma. However, the oncolytic capacity of SFV is restricted by the anti-viral type-I interferon (IFN) response. The aim of this study was to increase the oncolytic capacity of a microRNA target tagged SFV against glioblastoma by arming it with the Vaccinia-virus-encoded type-I IFN decoy receptor B18R (SFV4B18RmiRT) to neutralize type-I IFN response. Expression of B18R by SFV4B18RmiRT aided neutralization of IFN-b, which was shown by reduced STAT-1 phosphorylation and improved virus spread in plaque assays. B18R expression by SFV4 increased its oncolytic capacity in vitro against murine glioblastoma (CT-2A), regardless of the presence of exogenous IFN-b. Both SFV4B18RmiRT and SFV4miRT treatments controlled tumor growth in mice with syngeneic orthotopic gliomablastoma (CT-2A). However, treatment with SFV4B18RmiRT induced severe neurological symptoms in some mice because of virus replication in the healthy brain. Neither neurotoxicity nor virus replication in the brain was observed when SFV4miRT was administered. In summary, our results indicate that the oncolytic capacity of SFV4 was improved in vitro and in vivo by incorporation of B18R, but neurotoxicity of the virus was increased, possibly due to loss of microRNA targets.
|
|
| 9. |
|
|
| 10. |
|
|
