| 1. |
- Bestas, Burcu, et al.
(author)
-
Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
- 2014
-
In: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 124:9, s. 4067-4081
-
Journal article (peer-reviewed)abstract
- X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.
|
|
| 2. |
- Carlsson, Per-Ola, et al.
(author)
-
Umbilical cord-derived mesenchymal stromal cells preserve endogenous insulin production in type 1 diabetes : a Phase I/II randomised double-blind placebo-controlled trial
- 2023
-
In: Diabetologia. - : Springer. - 0012-186X .- 1432-0428. ; 66:8, s. 1431-1441
-
Journal article (peer-reviewed)abstract
- Aim/hypothesis This study aimed to investigate the safety and efficacy of treatment with allogeneic Wharton's jelly-derived mesenchymal stromal cells (MSCs) in recent-onset type 1 diabetes.Methods A combined Phase I/II trial, composed of a dose escalation followed by a randomised double-blind placebo-controlled study in parallel design, was performed in which treatment with allogeneic MSCs produced as an advanced therapy medicinal product (ProTrans) was compared with placebo in adults with newly diagnosed type 1 diabetes. Inclusion criteria were a diagnosis of type 1 diabetes <2 years before enrolment, age 18-40 years and a fasting plasma C-peptide concentration >0.12 nmol/l. Randomisation was performed with a web-based randomisation system, with a randomisation code created prior to the start of the study. The randomisation was made in blocks, with participants randomised to ProTrans or placebo treatment. Randomisation envelopes were kept at the clinic in a locked room, with study staff opening the envelopes at the baseline visits. All participants and study personnel were blinded to group assignment. The study was conducted at Karolinska University Hospital, Stockholm, Sweden.Results Three participants were included in each dose cohort during the first part of the study. Fifteen participants were randomised in the second part of the study, with ten participants assigned to ProTrans treatment and five to placebo. All participants were analysed for the primary and secondary outcomes. No serious adverse events related to treatment were observed and, overall, few adverse events (mainly mild upper respiratory tract infections) were reported in the active treatment and placebo arms. The primary efficacy endpoint was defined as Delta-change in C-peptide AUC for a mixed meal tolerance test at 1 year following ProTrans/placebo infusion compared with baseline performance prior to treatment. C-peptide levels in placebo-treated individuals declined by 47%, whereas those in ProTrans-treated individuals declined by only 10% (p<0.05). Similarly, insulin requirements increased in placebo-treated individuals by a median of 10 U/day, whereas insulin needs of ProTrans-treated individuals did not change over the follow-up period of 12 months (p<0.05).Conclusions/interpretation This study suggests that allogeneic Wharton's jelly-derived MSCs (ProTrans) is a safe treatment for recent-onset type 1 diabetes, with the potential to preserve beta cell function.
|
|
| 3. |
- EL Andaloussi, Samir, et al.
(author)
-
Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
- 2011
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:9, s. 3972-3987
-
Journal article (peer-reviewed)abstract
- While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.
|
|
| 4. |
- Gupta, Dhanu, et al.
(author)
-
Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles
- 2021
-
In: Nature Biomedical Engineering. - Stockholm : Karolinska Institutet, Dept of Laboratory Medicine. - 2157-846X.
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.
|
|
| 5. |
- Müller, Thomas R., et al.
(author)
-
Additive effects of booster mRNA vaccination and SARS-CoV-2 Omicron infection on T cell immunity across immunocompromised states
- 2023
-
In: Science Translational Medicine. - 1946-6234 .- 1946-6242. ; 15:704, s. eadg9452-
-
Journal article (peer-reviewed)abstract
- Suboptimal immunity to SARS-CoV-2 mRNA vaccination has frequently been observed in individuals with various immunodeficiencies. Given the increased antibody evasion properties of emerging SARS-CoV-2 subvariants, it is necessary to assess whether other components of adaptive immunity generate resilient and protective responses against infection. We assessed T cell responses in 279 individuals, covering five different immunodeficiencies and healthy controls, before and after booster mRNA vaccination, as well as after Omicron infection in a subset of patients. We observed robust and persistent Omicron-reactive T cell responses that increased markedly upon booster vaccination and correlated directly with antibody titers across all patient groups. Poor vaccination responsiveness in immunocompromised or elderly individuals was effectively counteracted by the administration of additional vaccine doses. Functionally, Omicron-reactive T cell responses exhibited a pronounced cytotoxic profile and signs of longevity, characterized by CD45RA+ effector memory subpopulations with stem cell-like properties and increased proliferative capacity. Regardless of underlying immunodeficiency, booster-vaccinated and Omicron-infected individuals appeared protected against severe disease and exhibited enhanced and diversified T cell responses against conserved and Omicron-specific epitopes. Our findings indicate that T cells retain the ability to generate highly functional responses against newly emerging variants, even after repeated antigen exposure and a robust immunological imprint from ancestral SARS-CoV-2 mRNA vaccination.
|
|
| 6. |
- Nordin, Joel Z., et al.
(author)
-
Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties
- 2015
-
In: Nanomedicine. - : Elsevier BV. - 1549-9634 .- 1549-9642. ; 11:4, s. 879-883
-
Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading toadifferent in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs.
|
|
| 7. |
- Schaafsma, Gerard C.P., et al.
(author)
-
BTKbase, Bruton Tyrosine Kinase Variant Database in X-Linked Agammaglobulinemia : Looking Back and Ahead
- 2023
-
In: Human Mutation. - 1059-7794. ; 2023
-
Journal article (peer-reviewed)abstract
- BTKbase is an international database for disease-causing variants in Bruton tyrosine kinase (BTK) leading to X-linked agammaglobulinemia (XLA), a rare primary immunodeficiency of antibody production. BTKbase was established in 1994 as one of the first publicly available variation databases. The number of cases has more than doubled since the last update; it now contains information for 2310 DNA variants in 2291 individuals. 1025 of the DNA variants are unique. The human genome contains more than 500 protein kinases, among which BTK has the largest number of unique disease-causing variants. The current version of BTKbase has numerous novel features: the database has been reformatted, it has moved to LOVD database management system, it has been internally harmonized, etc. Systematics and standardization have been increased, including Variation Ontology annotations for variation types. There are some regions with lower than expected variation frequency and some hotspots for variations. BTKbase contains, in addition to variant descriptions at DNA, RNA and protein levels, also laboratory parameters and clinical features for many patients. BTKbase has served clinical and research communities in the diagnosis of XLA cases and provides general insight into effects of variations, especially in signalling pathways. Amino acid substitutions and their effects were investigated, predicted, and visualized at 3D level in the protein domains. BTKbase is freely available.
|
|
| 8. |
- Bergman, Peter, et al.
(author)
-
Safety and efficacy of the mRNA BNT162b2 vaccine against SARS-CoV-2 in five groups of immunocompromised patients and healthy controls in a prospective open-label clinical trial
- 2021
-
In: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 74
-
Journal article (peer-reviewed)abstract
- Background: Patients with immunocompromised disorders have mainly been excluded from clinical trials of vaccination against COVID-19. Thus, the aim of this prospective clinical trial was to investigate safety and efficacy of BNT162b2 mRNA vaccination in five selected groups of immunocompromised patients and healthy controls.Methods: 539 study subjects (449 patients and 90 controls) were included. The patients had either primary (n=90), or secondary immunodeficiency disorders due to human immunodeficiency virus infection (n=90), allogeneic hematopoietic stem cell transplantation/CAR T cell therapy (n=90), solid organ transplantation (SOT) (n=89), or chronic lymphocytic leukemia (CLL) (n=90). The primary endpoint was seroconversion rate two weeks after the second dose. The secondary endpoints were safety and documented SARS-CoV-2 infection.Findings: Adverse events were generally mild, but one case of fatal suspected unexpected serious adverse reaction occurred. 72.2% of the immunocompromised patients seroconverted compared to 100% of the controls (p=0.004). Lowest seroconversion rates were found in the SOT (43.4%) and CLL (63.3%) patient groups with observed negative impact of treatment with mycophenolate mofetil and ibrutinib, respectively.Interpretation: The results showed that the mRNA BNT162b2 vaccine was safe in immunocompromised patients. Rate of seroconversion was substantially lower than in healthy controls, with a wide range of rates and antibody titres among predefined patient groups and subgroups. This clinical trial highlights the need for additional vaccine doses in certain immunocompromised patient groups to improve immunity.
|
|
| 9. |
- Bergquist, Helen, et al.
(author)
-
Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)(n) Repeats by PNA or LNA Targeting
- 2016
-
In: PLOS ONE. - : PLOS. - 1932-6203. ; 11:11
-
Journal article (peer-reviewed)abstract
- Expansion of (GAA)(n) repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich's ataxia. (GAA)(n) expansions form non-canonical structures, including intramolecular triplex (H-DNA), and Rloops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)(n) expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)(n) repeats of different lengths (short: n= 9, medium: n= 75 or long: n= 115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA) 4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT) 5-PNA did not. We present evidence that (GAA) 4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT) 5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.
|
|
| 10. |
|
|
| 11. |
- Bost, Jeremy P., et al.
(author)
-
Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides
- 2022
-
In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1
-
Journal article (peer-reviewed)abstract
- The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
|
|
| 12. |
- Brandén, Lars J., et al.
(author)
-
A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA
- 1999
-
In: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 17:8, s. 784-787
-
Journal article (peer-reviewed)abstract
- We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA–NLS peptide. The PNA–NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA–NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA–PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.
|
|
| 13. |
- Cardona Gomez, Maria Eugenia, et al.
(author)
-
Specific properties of shRNA-mediated CCR5 downregulation that enhance the inhibition of HIV-1 infection in combination with shRNA targeting HIV-1 rev
- 2022
-
In: Molecular Biology Reports. - : Springer. - 0301-4851 .- 1573-4978. ; 49, s. 11187-11192
-
Journal article (peer-reviewed)abstract
- Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.
|
|
| 14. |
- Chen, Puran, et al.
(author)
-
Real-world assessment of immunogenicity in immunocompromised individuals following SARS-CoV-2 mRNA vaccination : a one-year follow-up of the prospective clinical trial COVAXID
- 2023
-
In: EBioMedicine. - : Elsevier. - 2352-3964. ; 94
-
Journal article (peer-reviewed)abstract
- Background: Immunocompromised patients have varying responses to SARS-CoV-2 mRNA vaccination. However, there is limited information available from prospective clinical trial cohorts with respect to long-term immunogenicity-related responses in these patient groups following three or four vaccine doses, and in applicable cases infection.Methods: In a real-world setting, we assessed the long-term immunogenicity-related responses in patients with primary and secondary immunodeficiencies from the prospective open-label clinical trial COVAXID. The original clinical trial protocol included two vaccine doses given on days 0 and 21, with antibody titres measured at six different timepoints over six months. The study cohort has subsequently been followed for one year with antibody responses evaluated in relation to the third and fourth vaccine dose, and in applicable cases SARS-CoV-2 infection. In total 356/539 patients were included in the extended cohort. Blood samples were analysed for binding antibody titres and neutralisation against the Spike protein for all SARS-CoV-2 variants prevailing during the study period, including Omicron subvariants. SARS-CoV-2 infections that did not require hospital care were recorded through quarterly in-person, or phone-, interviews and assessment of IgG antibody titres against SARSCoV-2 Nucleocapsid. The original clinical trial was registered in EudraCT (2021-000175-37) and clinicaltrials.gov (NCT04780659).Findings: The third vaccine dose significantly increased Spike IgG titres against all the SARS-CoV-2 variants analysed in all immunocompromised patient groups. Similarly, neutralisation also increased against all variants studied, except for Omicron. Omicron-specific neutralisation, however, increased after a fourth dose as well as after three doses and infection in many of the patient subgroups. Noteworthy, however, while many patient groups mounted strong serological responses after three and four vaccine doses, comparably weak responders were found among patient subgroups with specific primary immunodeficiencies and subgroups with immunosuppressive medication.Interpretation: The study identifies particularly affected patient groups in terms of development of long-term immunity among a larger group of immunocompromised patients. In particular, the results highlight poor vaccine-elicited neutralising responses towards Omicron subvariants in specific subgroups. The results provide additional knowledge of relevance for future vaccination strategies.
|
|
| 15. |
- Cuapio, Angelica, et al.
(author)
-
NK cell frequencies, function and correlates to vaccine outcome in BNT162b2 mRNA anti-SARS-CoV-2 vaccinated healthy and immunocompromised individuals
- 2022
-
In: Molecular Medicine. - : BioMed Central (BMC). - 1076-1551 .- 1528-3658. ; 28:1
-
Journal article (peer-reviewed)abstract
- Adaptive immune responses have been studied extensively in the course of mRNA vaccination against COVID-19. Considerably fewer studies have assessed the effects on innate immune cells. Here, we characterized NK cells in healthy individuals and immunocompromised patients in the course of an anti-SARS-CoV-2 BNT162b2 mRNA prospective, open-label clinical vaccine trial. See trial registration description in notes. Results revealed preserved NK cell numbers, frequencies, subsets, phenotypes, and function as assessed through consecutive peripheral blood samplings at 0, 10, 21, and 35 days following vaccination. A positive correlation was observed between the frequency of NKG2C+ NK cells at baseline (Day 0) and anti-SARS-CoV-2 Ab titers following BNT162b2 mRNA vaccination at Day 35. The present results provide basic insights in regards to NK cells in the context of mRNA vaccination, and have relevance for future mRNA-based vaccinations against COVID-19, other viral infections, and cancer.Trial registration: The current study is based on clinical material from the COVAXID open-label, non-randomized prospective clinical trial registered at EudraCT and clinicaltrials.gov (no. 2021–000175-37). Description: https://clinicaltrials.gov/ct2/show/NCT04780659?term=2021-000175-37&draw=2&rank=1.
|
|
| 16. |
- Ezzat, Kariem, et al.
(author)
-
PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
- 2011
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:12, s. 5284-5298
-
Journal article (peer-reviewed)abstract
- Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.
|
|
| 17. |
- Ezzat, Kariem, et al.
(author)
-
Solid formulation of cell-penetrating peptide nanoparticles with siRNA and their stability in simulated gastric conditions
- 2012
-
In: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 162:1, s. 1-8
-
Journal article (peer-reviewed)abstract
- Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect was obtained at low siRNA doses with a unique kinetic profile. Furthermore, we utilized the solid dispersion technique to formulate PF14/siRNA nanocomplexes into solid formulations that were as active as the freshly prepared nanocomplexes in solution. Importantly, the freshly prepared nanocomplexes and solid formulations were stable after incubation with simulated gastric fluid having a pH of 1.2 and containing proteolytic enzymes. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.
|
|
| 18. |
- Gao, Yu, et al.
(author)
-
Immunodeficiency syndromes differentially impact the functional profile of SARS-CoV-2-specific T cells elicited by mRNA vaccination
- 2022
-
In: Immunity. - : Elsevier. - 1074-7613 .- 1097-4180. ; 55:9, s. 1732-1746.e5
-
Journal article (peer-reviewed)abstract
- Many immunocompromised patients mount suboptimal humoral immunity after SARS-CoV-2 mRNA vaccination. Here, we assessed the single-cell profile of SARS-CoV-2-specific T cells post-mRNA vaccination in healthy individuals and patients with various forms of immunodeficiencies. Impaired vaccine-induced cell-mediated immunity was observed in many immunocompromised patients, particularly in solid-organ transplant and chronic lymphocytic leukemia patients. Notably, individuals with an inherited lack of mature B cells, i.e., X-linked agammaglobulinemia (XLA) displayed highly functional spike-specific T cell responses. Single-cell RNA-sequencing further revealed that mRNA vaccination induced a broad functional spectrum of spike-specific CD4+ and CD8+ T cells in healthy individuals and patients with XLA. These responses were founded on polyclonal repertoires of CD4+ T cells and robust expansions of oligoclonal effector-memory CD45RA+ CD8+ T cells with stem-like characteristics. Collectively, our data provide the functional continuum of SARS-CoV-2-specific T cell responses post-mRNA vaccination, highlighting that cell-mediated immunity is of variable functional quality across immunodeficiency syndromes.
|
|
| 19. |
- Geny, Sylvain, et al.
(author)
-
Next-generation bis-locked nucleic acids with stacking linker and 2 '-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes
- 2016
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:5, s. 2007-2019
-
Journal article (peer-reviewed)abstract
- Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
|
|
| 20. |
- Hamada-Kawaguchi, Noriko, 1978-, et al.
(author)
-
Exposure to Therapeutic BTK Inhibitors Induces Phenocopying of Btk29A Mutants in the Fruit Fly Drosophila melanogaster
- 2023
-
In: Frontiers in Bioscience-Landmark. - 2768-6701 .- 2768-6698. ; 28:6
-
Journal article (peer-reviewed)abstract
- Background: Bruton’s tyrosine kinase (BTK) is a non-receptor type tyrosine kinase originally identified as the genetic signature responsible for X-linked agammaglobulinemia (XLA) when mutated. Its functional form is required for B lymphocyte maturation in both humans and mice, whereas loss-of-function causes a different form of developmental defect in the fruit fly, Drosophila melanogaster. Methods: Ibrutinib and other therapeutic inhibitors of BTK have been extensively used to successfully treat various leukemias and lymphomas. Btk29A type 2 is the ortholog of BTK in the fruit fly. We show that feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants, i.e., failure in the fusion of left and right halves of the dorsal cuticles, partial loss of wing tissues and dysregulation of germ cell production. Results: We have previously reported that Btk29A phosphorylates Drosophila Arm (β?-catenin), and ibrutinib reduces phosphorylation at Tyrosine142 of endogenously expressed β?-catenin in Cos7 cells transfected with Btk29A type 2 cDNA. Conclusions: Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.
|
|
| 21. |
- Hammarström, Lennart, et al.
(author)
-
Transfer of IgA deficiency to a bone-marrow-grafted patient with aplastic anaemia
- 1985
-
In: The Lancet. - 1474-547X. ; 325:8432, s. 778-781
-
Journal article (peer-reviewed)abstract
- IgA deficiency developed in a 2-year-old boy with aplastic anaemia who received a bone-marrow graft from his HLA-identical, 6-year-old, IgA-deficient sister. Southern blot analysis revealed the presence of alpha-genes in both children, thus suggesting a defect of lymphocyte stem-cell differentiation as a cause of IgA deficiency. Tissue typing showed homozygosity of HLA A1, B8, DR3, the haplotype associated with IgA deficiency in healthy people. Despite normal serum levels of IgG subclasses in both donor and recipient, both children showed a relative lack of specific IgG2 anticarbohydrate antibodies. This suggests that their IgA deficiency is part of a more fundamental aberration of immunoglobulin class and subclass distribution.
|
|
| 22. |
- Healy, Katie, et al.
(author)
-
Salivary IgG to SARS-CoV-2 indicates seroconversion and correlates to serum neutralization in mRNA-vaccinated immunocompromised individuals
- 2022
-
In: Med. - : Cell Press. - 2666-6359 .- 2666-6340. ; 3:2, s. 137-153.e3
-
Journal article (peer-reviewed)abstract
- Background: Immunocompromised individuals are highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Whether vaccine-induced immunity in these individuals involves oral cavity, a primary site of infection, is presently unknown.Methods: Immunocompromised patients (n = 404) and healthy controls (n = 82) participated in a prospective clinical trial (NCT04780659) encompassing two doses of the mRNA BNT162b2 vaccine. Primary immunodeficiency (PID), secondary immunodeficiencies caused by human immunodeficiency virus (HIV) infection, allogeneic hematopoietic stem cell transplantation (HSCT)/chimeric antigen receptor T cell therapy (CAR-T), solid organ transplantation (SOT), and chronic lymphocytic leukemia (CLL) patients were included. Salivary and serum immunoglobulin G (IgG) reactivities to SARS-CoV-2 spike were measured by multiplex bead-based assays and Elecsys anti-SARS-CoV-2 S assay.Findings: IgG responses to SARS-CoV-2 spike antigens in saliva in HIV and HSCT/CAR-T groups were comparable to those of healthy controls after vaccination. The PID, SOT, and CLL patients had weaker responses, influenced mainly by disease parameters or immunosuppressants. Salivary responses correlated remarkably well with specific IgG titers and the neutralizing capacity in serum. Receiver operating characteristic curve analysis for the predictive power of salivary IgG yielded area under the curve (AUC) = 0.95 and positive predictive value (PPV) = 90.7% for the entire cohort after vaccination.Conclusions: Saliva conveys vaccine responses induced by mRNA BNT162b2. The predictive power of salivary spike IgG makes it highly suitable for screening vulnerable groups for revaccination.
|
|
| 23. |
|
|
| 24. |
- Jarviaho, Tekla, et al.
(author)
-
Predisposition to childhood acute lymphoblastic leukemia caused by a constitutional translocation disrupting ETV6
- 2019
-
In: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 3:18, s. 2722-2731
-
Journal article (peer-reviewed)abstract
- Pathogenic germline variants in ETV6 have been associated with familial predisposition to thrombocytopenia and hematological malignancies, predominantly childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In addition, overrepresentation of a high hyperdiploid subtype and older age at diagnosis have been reported among sporadic BCP-ALL cases with germline variants in ETV6. We studied a family with 2 second-degree relatives who developed childhood high hyperdiploid BCP-ALL at ages 8 and 12 years, respectively. A constitutional balanced reciprocal translocation t(12;14)(p13.2;q23.1) was discovered in both patients by routine karyotyping at diagnosis and, subsequently, in 7 healthy family members who had not experienced hematological malignancies. No carriers had thrombocytopenia. Whole-genome sequencing confirmed the translocation, resulting in 2 actively transcribed but nonfunctional fusion genes, causing heterozygous loss and consequently monoallelic expression of ETV6. Whole-genome sequencing analysis of the affected female subjects' leukemia excluded additional somatic aberrations in ETV6 and RTN1 as well as shared somatic variants in other genes. Expression studies, performed to confirm decreased expression of ETV6, were not conclusive. We suggest that germline aberrations resulting in monoallelic expression of ETV6 contribute to leukemia susceptibility, whereas more severe functional deficiency of ETV6 is required for developing THC5. To our knowledge, this report is the first of a constitutional translocation disrupting ETV6 causing predisposition to childhood ALL.
|
|
| 25. |
- Järver, Peter, et al.
(author)
-
Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.
- 2015
-
In: Nucleic Acid Therapeutics. - : Mary Ann Liebert Inc. - 2159-3337 .- 2159-3345. ; 25:2, s. 65-77
-
Journal article (peer-reviewed)abstract
- Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.
|
|
| 26. |
- Lehto, Taavi, et al.
(author)
-
A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
- 2011
-
In: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 19:8, s. 1457-1467
-
Journal article (peer-reviewed)abstract
- Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.
|
|
| 27. |
- Lindahl, Hannes, et al.
(author)
-
SARS-CoV-2 Antibodies in Commercial Immunoglobulin Products Show Markedly Reduced Cross-reactivities Against Omicron Variants
- 2023
-
In: Journal of Clinical Immunology. - : Springer Nature. - 0271-9142 .- 1573-2592. ; 43:6, s. 1075-1082
-
Journal article (peer-reviewed)abstract
- PurposePatients with antibody deficiencies often receive maintenance treatment with donor plasma-derived immunoglobulin (Ig) preparations to decrease the incidence and severity of infections. We have previously shown that IgG antibodies to the original SARS-CoV-2 strain were not consistently present in off-the-shelf Ig batches produced up to approximately 18 months after the first identified case of COVID-19 in the USA and that Ig batches with anti-SARS-CoV-2 IgG primarily contained vaccine-induced spike specific antibodies. This study aimed to investigate the degree of cross-reactivity between vaccine-induced anti-SARS-CoV-2 antibodies against Wuhan strain and subsequent viral variants.MethodsSamples were collected from 74 Ig batches supplied by three different commercial manufacturers. All batches were used at the Immunodeficiency Unit at the Karolinska University Hospital from the start of the SARS-CoV-2 pandemic until September 2022. Antibody quantity and potential to neutralize virus entry into host cells were assessed against the original SARS-CoV-2 Wuhan strain and the following nine variants: Alpha, Beta, Delta, IHU, and the Omicron BA.1, BA.1.1, BA.1 with spike mutation L452R, BA.2, and BA.3.ResultsIg batches produced approximately 18 months after the SARS-CoV-2 outbreak (from around July 2021) and later consistently contained high quantities of antibodies that bind the Wuhan strain. The Ig batches had overall low reactivity to the SARS-CoV-2 nucleocapsid, which implies that plasma donor spike IgG essentially is the result of vaccination. We assessed the degree of cross-reactivity towards each virus variant by plotting the variant/Wuhan strain ratio, which was consistent regardless of production date, suggesting cross-reactivity with vaccine-induced antibodies rather than virus exposure in the plasma donor population. Viral variants that emerged later during the pandemic systematically had a lower reactivity ratio, except for the Delta and IHU variants. The Ig batches displayed markedly low neutralizing potential towards the Beta variant and all tested Omicron variants.ConclusionCommercial Ig batches currently contain large quantities of SARS-CoV-2 vaccine-induced antibodies. Cross-reactivity with variant strains is evident but varies, with markedly low neutralizing potential observed against Omicron variants.
|
|
| 28. |
- Lundin, Karin E., et al.
(author)
-
Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report
- 2018
-
In: BMC Pediatrics. - : Springer. - 1471-2431. ; 18
-
Journal article (peer-reviewed)abstract
- Background: A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 +3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. Case presentation: We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naive CD4(+) and CD8(+) T-cells, increased number of activated effector memory CD8(+) T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient's cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. Conclusions: There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing.
|
|
| 29. |
- Lundin, Karin E, et al.
(author)
-
Susceptibility to infections, without concomitant hyper-IgE, reported in 1976, is caused by hypomorphic mutation in the phosphoglucomutase 3 (PGM3) gene
- 2015
-
In: Clinical Immunology. - : Elsevier. - 1521-6616 .- 1521-7035. ; 161:2, s. 366-372
-
Journal article (peer-reviewed)abstract
- Phosphoglucomutase 3 (PGM3) is an enzyme converting N-acetyl-glucosamine-6-phosphate to N-acetylglucosamine-l-phosphate, a precursor important for glycosylation. Mutations in the PGM3 gene have recently been identified as the cause of novel primary immunodeficiency with a hyper-IgE like syndrome. Here we report the occurrence of a homozygous mutation in the PGM3 gene in a family with immunodeficient children, described already in 1976. DNA from two of the immunodeficient siblings was sequenced and shown to encode the same homozygous missense mutation, causing a destabilized protein with reduced enzymatic capacity. Affected individuals were highly prone to infections, but lack the developmental defects in the nervous and skeletal systems, reported in other families. Moreover, normal IgE levels were found. Thus, belonging to the expanding group of congenital glycosylation defects, PGM3 deficiency is characterized by immunodeficiency, with or without increased IgE levels, and with variable forms of developmental defects affecting other organ systems.
|
|
| 30. |
- Moens, Lotte N., et al.
(author)
-
Diagnostics of Primary Immunodeficiency Diseases : A Sequencing Capture Approach
- 2014
-
In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:12, s. e114901-
-
Journal article (peer-reviewed)abstract
- Primary Immunodeficiencies (PID) are genetically inherited disorders characterized by defects of the immune system, leading to increased susceptibility to infection. Due to the variety of clinical symptoms and the complexity of current diagnostic procedures, accurate diagnosis of PID is often difficult in daily clinical practice. Thanks to the advent of next generation'' sequencing technologies and target enrichment methods, the development of multiplex diagnostic assays is now possible. In this study, we applied a selector-based target enrichment assay to detect disease-causing mutations in 179 known PID genes. The usefulness of this assay for molecular diagnosis of PID was investigated by sequencing DNA from 33 patients, 18 of which had at least one known causal mutation at the onset of the experiment. We were able to identify the disease causing mutations in 60% of the investigated patients, indicating that the majority of PID cases could be resolved using a targeted sequencing approach. Causal mutations identified in the unknown patient samples were located in STAT3, IGLL1, RNF168 and PGM3. Based on our results, we propose a stepwise approach for PID diagnostics, involving targeted resequencing, followed by whole transcriptome and/or whole genome sequencing if causative variants are not found in the targeted exons.
|
|
| 31. |
- Mohammad, Dara K., et al.
(author)
-
B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing
- 2016
-
In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:8
-
Journal article (peer-reviewed)abstract
- Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins.
|
|
| 32. |
- Müller, Thomas R., et al.
(author)
-
Memory T cells effectively recognize the SARS-CoV-2 hypermutated BA.2.86 variant
- 2024
-
In: Cell Host and Microbe. - : Elsevier. - 1931-3128 .- 1934-6069. ; 32:2, s. 156-161.e3
-
Journal article (peer-reviewed)abstract
- T cells are critical in mediating the early control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection. However, it remains unknown whether memory T cells can effectively cross-recognize new SARS-CoV-2 variants with a broad array of mutations, such as the emergent hypermutated BA.2.86 variant. Here, we report in two separate cohorts, including healthy controls and individuals with chronic lymphocytic leukemia, that SARS-CoV-2 spike-specific CD4+ and CD8+ T cells induced by prior infection or vaccination demonstrate resilient immune recognition of BA.2.86. In both cohorts, we found largely preserved SARS-CoV-2 spike-specific CD4+ and CD8+ T cell magnitudes against mutated spike epitopes of BA.2.86. Functional analysis confirmed that both cytokine expression and proliferative capacity of SARS-CoV-2 spike-specific T cells to BA.2.86-mutated spike epitopes are similarly sustained. In summary, our findings indicate that memory CD4+ and CD8+ T cells continue to provide cell-mediated immune recognition to highly mutated emerging variants such as BA.2.86.
|
|
| 33. |
- Naeem, Aishath, et al.
(author)
-
Pirtobrutinib targets BTK C481S in ibrutinib-resistant CLL but second-site BTK mutations lead to resistance
- 2023
-
In: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 7:9, s. 1929-1943
-
Journal article (peer-reviewed)abstract
- Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKis) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, noncovalent BTKi with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and cells from patients with CLL, we show that pirtobrutinib potently inhibits BTK-mediated functions including B-cell receptor (BCR) signaling, cell viability, and CCL3/CCL4 chemokine production in both BTK wild-type and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival, and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability, and cytokine production. We employed longitudinal whole-exome sequencing on 2 patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to noncovalent BTKis.
|
|
| 34. |
- Nore, Beston F, et al.
(author)
-
Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases
- 2003
-
In: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1645:2, s. 123-132
-
Journal article (peer-reviewed)abstract
- Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton's tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.
|
|
| 35. |
- Palma, Marzia, et al.
(author)
-
Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes
- 2018
-
In: British Journal of Haematology. - : WILEY. - 0007-1048 .- 1365-2141. ; 183:2, s. 212-224
-
Journal article (peer-reviewed)abstract
- In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19(+) circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.
|
|
| 36. |
|
|
| 37. |
- Sassi, Atfa, et al.
(author)
-
Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels
- 2014
-
In: Journal of Allergy and Clinical Immunology. - : Elsevier BV. - 0091-6749 .- 1097-6825. ; 133:5, s. 1410-U681
-
Journal article (peer-reviewed)abstract
- Background: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. Objective: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. Methods: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. Results: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p. Glu340del and p. Leu83Ser). A third homozygous mutation (p. Asp502Tyr) and the p. Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. Conclusion: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.
|
|
| 38. |
- Smith, C. I Edvard, et al.
(author)
-
Genetics of Immune Disease
- 1992
-
In: Tissue Antigens. - : Wiley. - 0001-2815 .- 1399-0039. ; 39:3, s. 159-159
-
Journal article (peer-reviewed)
|
|
| 39. |
- Sork, Helena, et al.
(author)
-
Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency
- 2016
-
In: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 5
-
Journal article (peer-reviewed)abstract
- The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
|
|
| 40. |
|
|
| 41. |
- Svahn, Mathias G., et al.
(author)
-
Self-assembling supramolecular complexes by single-stranded extension from plasmid DNA
- 2007
-
In: Oligonucleotides. - : Mary Ann Liebert Inc. - 1545-4576 .- 1557-8526. ; 17:1, s. 80-94
-
Journal article (peer-reviewed)abstract
- Self-assembling supramolecular complexes are of great interest for bottom-up research like nanotechnology. DNA is an inexpensive building block with sequence-specific self-assembling capabilities through Watson-Crick and/or Hoogsteen base pairing and could be used for applications in surface chemistry, material science, nanomechanics, nanoelectronics, nanorobotics, and of course in biology. The starting point is usually single-stranded DNA, which is rather easily accessible for base pairing and duplex formation. When long stretches of double-stranded DNA are desirable, serving either as genetic codes or electrical wires, bacterial expansion of plasmids is an inexpensive approach with scale-up properties. Here, we present a method for using double-stranded DNA of any sequence for generating simple structures, such as junctions and DNA lattices. It is known that supercoiled plasmids are strand-invaded by certain DNA analogs. Here we add to the complexity by using Selfassembling UNiversal (SUN) anchors formed by DNA analog oligonucleotides, synthesized with an extension, a sticky-end that can be used for further base pairing with single-stranded DNA. We show here how the same set of SUN anchors can be utilized for gene therapy, plasmid purification, junction for lattices, and plasmid dimerization through Watson-Crick base pairing. Using atomic force microscopy, it has been possible to characterize and quantify individual components of such supra-molecular complexes.
|
|
| 42. |
- Umek, Tea, et al.
(author)
-
Oligonucleotide Binding to Non-B-DNA in MYC
- 2019
-
In: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 24:5
-
Journal article (peer-reviewed)abstract
- MYC, originally named c-myc, is an oncogene deregulated in many different forms of cancer. Translocation of the MYC gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt's lymphoma (BL). Sporadic BL constitutes one subgroup where one of the translocation sites is located at the 5'-vicinity of the two major MYC promoters P-1 and P-2. A non-B-DNA forming sequence within this region has been reported with the ability to form an intramolecular triplex (H-DNA) or a G-quadruplex. We have examined triplex formation at this site first by using a 17 bp triplex-forming oligonucleotide (TFO) and a double strand DNA (dsDNA) target corresponding to the MYC sequence. An antiparallel purine-motif triplex was detected using electrophoretic mobility shift assay. Furthermore, we probed for H-DNA formation using the BQQ-OP based triplex-specific cleavage assay, which indicated the formation of the structure in the supercoiled plasmid containing the corresponding region of the MYC promoter. Targeting non-B-DNA structures has therapeutic potential; therefore, we investigated their influence on strand-invasion of anti-gene oligonucleotides (ON)s. We show that in vitro, non-B-DNA formation at the vicinity of the ON target site facilitates dsDNA strand-invasion of the anti-gene ONs.
|
|
| 43. |
- Valiaho, Jouni, et al.
(author)
-
Characterization of All Possible Single-Nucleotide Change Caused Amino Acid Substitutions in the Kinase Domain of Bruton Tyrosine Kinase
- 2015
-
In: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 36:6, s. 638-647
-
Journal article (peer-reviewed)abstract
- Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease-causing variations among the human protein kinases, still it is just 10% of all the possible single-nucleotide substitution-caused amino acid variations (SNAVs). In the BTK kinase domain (BTK-KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease-causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty-seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms.
|
|
| 44. |
- Westerlind, Ulrika, et al.
(author)
-
Ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: Synthesis of and binding studies with biotinylated cluster glycosides containing N-acetylgalactosamine.
- 2004
-
In: Glycoconjugate journal. - : Springer. - 0282-0080 .- 1573-4986. ; 21:5, s. 227-41
-
Journal article (peer-reviewed)abstract
- In order to develop the non-viral Bioplex vector system for targeted delivery of genes to hepatocytes, we have evaluated the structure-function relationship for a number of synthetic ligands designed for specific interaction with the hepatic lectin ASGPr. Biotinylated ligand derivatives containing two, three or six beta-linked N-acetylgalactosamine (GalNAc) residues were synthesized, bound to fluorescent-labeled streptavidin and tested for binding and uptake to HepG2 cells using flow cytometry analysis (FACS). Uptake efficiency increased with number of displayed GalNAc units per ligand, in a receptor dependent manner. Thus, a derivative displaying six GalNAc units showed the highest uptake efficacy both in terms of number of internalizing cells and increased amount of material taken up per each cell. However, this higher efficiency was shown to be due not so much to higher number of sugar units, but to higher accessibility of the sugar units for interaction with the receptor (longer spacer). Improving the flexibility and accessibility of a trimeric GalNAc ligand through use of a longer spacer markedly influenced the uptake efficiency, while increasing the number of GalNAc units per ligand above three only provided a minor contribution to the overall affinity. We hereby report the details of the chemical synthesis of the ligands and the structure-function studies in vitro.
|
|
| 45. |
- Zaghloul, Eman M., et al.
(author)
-
CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression
- 2017
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 45:9, s. 5153-5169
-
Journal article (peer-reviewed)abstract
- Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.
|
|
