Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A.

nefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking

CD133-targeted Gene Transfer Into Long-term Repopulating Hematopoietic Stem Cells

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells

Rescue of mutant FVIII proteins <i>in vitro</i> and <i>in vivo</i>.

<p>(A–D) HepG2 cells expressing hFVIII muteins were incubated with CC for 72 h. Amount of hFVIII activity in cell supernatant of HepG2 cells expressing hFVIII-BDD Q305P (A and C) or hFVIII-BDD W2313A (B and C) was measured 72 h post betaine treatment. (A and B) show the supplementation of single CC and (C) betaine-ectoine combined treatment. (D) Post CC incubation HepG2 hFVIII-BDDQ305P cells were successively lysed in PBS/0.5% Triton X-100 and PBS/1% SDS. hFVIII antigen was determined in both fractions by indirect ELISA. (E–J) hFVIII-BDDQ305P injected Hem A mice were treated with 2% betaine ad libitum per os in a crossover-study of two groups (each n = 10). After 3 days of treatment hFVIII antigen and activity was measured and treatment was switched between mouse groups. 3 days later, plasma levels were tested again. (E and F) show hFVIII antigen levels, (H and I) the related hFVIII activity levels in plasma of group I or II. (G and J) represent the calculated overall effect of betaine on FVIII antigen levels (G) or FVIII activity (J). square symbols indicate samples of the first measuring point, triangles the second one; clear: tap water-administration (control), filled: 2% betaine administration. (K and L) Endogenous murine FIX levels in all injected FVIII knockout mice (K) and murine FVIII levels of all used FIX knockout mice (L) with and without betaine in the drinking water. Normal mouse levels were set to 100%. Values are presented as means ± SEM. (A–D) ANOVA; (E–H; K–L) Student’s t-test; (I–J) Wilcoxon signed rank test;*<i>P</i><.05, **<i>P</i><.005, ***<i>P</i><.0005.</p

Betaine increases solubility of intracellular FVIII.

<p>CHO cells expressing eGFP-tagged FVIII-BDD protein were incubated with and without betaine. (A,B) Flow cytometry analysis was used to determine the eGFP-signal in untreated cells versus cells treated with betaine or control substance BHA. The mean eGFP intensity (X mean GFP) in the range M1 was used as distinctive parameter. (A) representative histogram after 48 h of treatment and (B) values after 72 h presented as means ± SEM of 3 independent experiments. ANOVA **<i>P</i><.001. (C,D) After 72 h incubation cells were successively lysed in PBS/0.5% Triton X-100 and PBS/1% SDS. (C) FVIII antigen was determined in both fractions by indirect ELISA. (D) Triton X-100-soluble and insoluble fractions were separated on SDS-polyacrylamid gradient gels, and hFVIII light chains (lc), eGFP in hFVIII-single chain (sc) and GAPDH were detected by Western blot. Δ indicates lower band of hFVIII lc doublet.</p

Betaine feeding improves hFVIII and hFIX secretion <i>in vivo</i>.

<p>(A–F) 24 hours post minicircle FVIII-BDD gene transfer the FVIII knockout mice received water without (group I) or with 2% betaine supplementation in the drinking water (group II, each n = 10). After 3 days plasma samples were collected and each group was monitored for human FVIII antigen (A and B) and related activity levels (D and E) and group treatment was switched. After another 3 days, plasma levels were tested again. (C and F) represent the calculated overall effect of betaine on FVIII antigen levels (C) or FVIII activity (D). square symbols indicate samples of the first measuring point, triangles the second one. (G–I) After reaching stable FIX expression levels following minicircle FIX gene transfer, FIX knockout mice were fed 2% Betaine-supplemented drinking water ad libitum in a crossover-study of two groups. After 3 and 17 days of treatment, retroorbitally collected plasma samples were monitored for human FIX antigen levels (G and H). (I) shows the overall change in FIX expression from both groups after 17 days of administration. All values are represented as mean ± SEM. Same symbols indicate samples of the same mouse at different time points; clear: tap water treatment (control), filled: betaine administration. Student’s t-test ((G) ANOVA). *<i>P</i><.05, **<i>P</i><.005, ***<i>P</i><.0005.</p

CC improve secretion of FVIII-BDD, FVIII-BDD-eGFP and FVIII-FL <i>in vitro</i>.

<p>Heterologous CHO cells were incubated with CC at different concentrations. FVIII activity was determined in cell supernatants after 72 h by chromogenic assay. (A) Effect of the following CC on human (h)FVIII-BDD secretion: Betaine (100; 50; 25 mM), ectoine (150; 100; 50 mM), trehalose (150; 100; 50 mM), sorbitol (150; 100; 50 mM), taurine (150; 100; 50 mM), trimethylamine N-oxide (TMAO;50; 25; 12,5 mM) and sodium 4-phenylbutyrate (4-PBA; 2; 0,4 mM). Number of experiments, n = 2. (B) Effect of betaine, ectoine, and the endoplasmatic ATPase inhibitors curcumin and thapsigargin on FVIII-.BDD-eGFP secretion. Butylated hydroxyanisole (BHA) is added as treatment control. n = 3. The mean FVIII secretion level ± SD of untreated hFVIII-BDD-eGFP expressing cells was 19±12 IU per 10e6 cells per 72 h. (C) FVIII-BDD-eGFP secretion into cell supernatants over time at different betaine concentrations. n = 3. (D) Influence of betaine, ectoine, curcumin and thapsigargin on FVIII-FL secretion 72 hours following drug supplementation. n = 3. All values are presented as means ± SEM. ANOVA test * <i>P</i><.05; ** <i>P</i><.001.</p

Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease

BACKGROUND: Sickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising alpha- and gamma-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of gamma-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF.METHODS: We enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage-specific knockdown. Patients were assessed for primary end points of engraftment and safety and for hematologic and clinical responses to treatment.RESULTS: As of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.5%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period.CONCLUSIONS: This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk-benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656)