Severe inflammation and lineage skewing are associated with poor engraftment of engineered hematopoietic stem cells in patients with sickle cell disease.

In sickle cell disease (SCD), the β6 substitution in the β-globin leads to red blood cell sickling. The transplantation of autologous, genetically modified hematopoietic stem and progenitor cells (HSPCs) is a promising treatment option for patients with SCD. We completed a Phase I/II open-label clinical trial (NCT03964792) for patients with SCD using a lentiviral vector (DREPAGLOBE) expressing a potent anti-sickling β-globin.

Genetic reversal of the globin switch concurrently modulates both fetal and sickle hemoglobin and reduces red cell sickling.

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications.

Long-term outcomes of lentiviral gene therapy for the β-hemoglobinopathies: the HGB-205 trial.

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34 cells transduced ex vivo with lentiviral vector BB305 that encodes the anti-sickling β-globin expressed in the erythroid lineage. HGB-205 is a phase 1/2, open-label, single-arm, non-randomized interventional study of 2-year duration at a single center, followed by observation in long-term follow-up studies LTF-303 ( NCT02633943 ) and LTF-307 ( NCT04628585 ) for TDT and SCD, respectively.

Correcting photodetector nonlinearity in dual-comb interferometry.

Photodetector nonlinearity, the main limiting factor in terms of optical power in the detection chain, is corrected to improve the signal-to-noise ratio of a short-time measurement in dual-comb spectroscopy. An iterative correction algorithm minimizing out-of-band spectral artifacts based on nonlinearity correction methods used in classical Fourier-transform spectrometers is presented. The exactitude of the nonlinearity correction is validated using a low power linear measurement.

Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease.

Polymerization of the sickle hemoglobin (HbS) is a key determinant of sickle cell disease (SCD), an inherited blood disorder. Fetal hemoglobin (HbF) is a major modulator of the disease severity by both decreasing HbS intracellular concentration and inhibiting its polymerization. However, heterocellular distribution of HbF is common in SCD.

Dual electro-optic frequency comb spectroscopy using pseudo-random modulation.

An approach for dual-comb spectroscopy using electro-optic (EO) phase modulation is reported. Maximum-length pseudo-random binary sequences allow for energy-efficient and flexible comb generation. Self-correction of interferograms is shown to remove relative comb drifts and improve mutual coherence, even for EO combs derived from the same laser source.

Methane spectroscopy using a free-running chip-based dual-comb laser.

Absorption lines of methane in the 2ν band centered at 1650 nm were measured with a free-running mode-locked dual-comb laser based on a single erbium-doped glass chip. The laser's spectra were broadened up to 1670 nm using amplifiers and highly nonlinear fiber. A comb was used to interrogate the complex transmission spectrum of a methane-filled gas cell with an optical point spacing of 968 MHz and an interferogram (IGM) rate of 27 kHz to yield absorption lines of the R and Q branches.

Highly coherent free-running dual-comb chip platform.

We characterize the frequency noise performance of a free-running dual-comb source based on an erbium-doped glass chip running two adjacent mode-locked waveguide lasers. This compact laser platform, contained only in a 1.2 L volume, rejects common-mode environmental noise by 20 dB thanks to the proximity of the two laser cavities.

Self-corrected chip-based dual-comb spectrometer.

We present a dual-comb spectrometer based on two passively mode-locked waveguide lasers integrated in a single Er-doped ZBLAN chip. This original design yields two free-running frequency combs having a high level of mutual stability. We developed in parallel a self-correction algorithm that compensates residual relative fluctuations and yields mode-resolved spectra without the help of any reference laser or control system.

Ultrafast pulse generation in a mode-locked Erbium chip waveguide laser.

We report mode-locked ~1550 nm output of transform-limited ~180 fs pulses from a large mode-area (diameter ~50 μm) guided-wave erbium fluorozirconate glass laser. The passively mode-locked oscillator generates pulses with 25 nm bandwidth at 156 MHz repetition rate and peak-power of 260 W. Scalability to higher repetition rate is demonstrated by transform-limited 410 fs pulse output at 1.

Dual-comb spectroscopy with a phase-modulated probe comb for sub-MHz spectral sampling.

We present a straightforward and efficient method to reduce the mode spacing of a frequency comb based on binary pseudo-random phase modulation of its pulse train. As a proof of concept, we use such a densified comb to perform dual-comb spectroscopy of a long-delay Mach-Zehnder interferometer and a high-quality-factor microresonator with sub-MHz spectral sampling. Since this approach is based on binary phase modulation, it combines all the advantages of other densification techniques: simplicity, single-step implementation, and conservation of the initial comb's power.

Complex direct comb spectroscopy with a virtually imaged phased array.

We demonstrate a simple interferometric technique to directly measure the complex optical transmittance over a large spectral range using a frequency-comb spectrometer based on a virtually imaged phased array. A Michelson interferometer encodes the phase deviations induced by a sample contained in one of its arms into an interferogram image. When combined with an additional image taken from each arm separately, along with a frequency-calibration image, this allows full reconstruction of the sample's optical transfer function.

Self-heterodyne interference spectroscopy using a comb generated by pseudo-random modulation.

We present an original instrument designed to accomplish high-speed spectroscopy of individual optical lines based on a frequency comb generated by pseudo-random phase modulation of a continuous-wave (CW) laser. This approach delivers efficient usage of the laser power as well as independent control over the spectral point spacing, bandwidth and central wavelength of the comb. The comb is mixed with a local oscillator generated from the same CW laser frequency-shifted by an acousto-optic modulator, enabling a self-heterodyne detection scheme.

A quantitative mode-resolved frequency comb spectrometer.

We have developed a frequency-comb spectrometer that records 35-nm (4 THz) spectra with 2-pm (250 MHz) spectral sampling and an absolute frequency accuracy of 2 kHz. We achieve a signal-to-noise ratio of ~400 in a measurement time of 8.2 s.

Molecular signature of erythroblast enucleation in human embryonic stem cells.

While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies.

Coherent dual-comb interferometry with quasi-integer-ratio repetition rates.

We demonstrate a generalized method for dual-comb interferometry that involves the use of two frequency combs with quasi-integer-ratio repetition rates. We use a 16.67 MHz comb to probe an 80-cm-long ring cavity and a 100 MHz comb to asynchronously sample its impulse response.

In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders.

Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs.

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model.

Background: Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors.

Design And Methods: We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin.

Proof of principle for transfusion of in vitro-generated red blood cells.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans.

Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine.

Background: Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine.