reblog

On December 8th, the U.S. Food and Drug Administration (FDA) approved two groundbreaking therapeutic drugs, Casgevy (developed jointly by Vertex Pharmaceuticals and CRISPR Therapeutics) and Lyfgenia (developed by Blue Bird Bio). These drugs represent the first wave of cell-based gene therapies approved by the FDA for the treatment of sickle cell disease (SCD) in patients aged 12 and above. Notably, Casgevy is the first FDA-approved therapeutic drug utilizing CRISPR gene editing technology, marking a significant milestone in the field of gene therapy.

Indications of Casgevy and Lyfgenia:

1. Casgevy:
   • Indication: Treatment for patients aged 12 and above with recurrent vaso-occlusive crises (VOCs) in sickle cell anemia (SCD).
2. Lyfgenia:
   • Indication: Treatment for patients aged 12 and above with a history of vaso-occlusive events (VOEs) in sickle cell disease.

It’s worth mentioning that Lyfgenia’s parent company, Blue Bird Bio, received approval for another drug called Zynteglo in August 2022, intended for the treatment of transfusion-dependent β-thalassemia (TDT).

Pathogenesis and Therapeutic Mechanism:

The structural composition of human hemoglobin consists of a heterotetramer, composed of two pairs of globin proteins. SCD and TDT result from variations in the β-globin protein. SCD involves a mutation where Glu6 in β-globin is replaced by Val6, causing hemoglobin to form long chains through hydrophobic interactions, leading to sickle-shaped cells and reduced oxygen-carrying capacity. TDT results from deficient synthesis of the β-globin chain, causing an excess of unbound α-chains, leading to precipitation and hemolysis within red blood cells.

• Casgevy’s Strategy:
  • Utilizes CRISPR technology to knock out the BCL11A gene.
  • BCL11A is a transcription factor that suppresses the expression of fetal hemoglobin (HbF).
  • Knocking out BCL11A results in upregulation of γ-globin expression and downregulation of β-globin, leading to the production of functional fetal hemoglobin (HbF).

• Lyfgenia’s Strategy:
  • Uses a lentivirus to insert an enhanced β-globin (T87Q) into the patient’s hematopoietic stem cells.
  • Generates hemoglobin (HbAT87Q) with anti-sickling properties, reducing the occurrence of VOEs.
  • HbAT87Q exhibits oxygen-carrying capacity similar to wild-type hemoglobin.

Both treatments involve extracting bone marrow hematopoietic stem cells from the patient, editing them in the laboratory using either CRISPR or viral vectors, and then reintroducing the edited stem cells into the patient.

Why (Only) Milestones:

While Casgevy and Lyfgenia represent significant milestones in the treatment of SCD, certain factors limit them from being the ultimate solutions. Safety concerns, potential carcinogenic risks associated with viral vectors (as in the case of Lyfgenia), and the long-term safety of gene editing are issues that need further investigation. Both therapies also involve myeloablation (bone marrow conditioning), leading to immune and reproductive challenges.

Another critical factor is the pricing. With Casgevy priced at $2.2 million and Lyfgenia at $3.1 million, the high costs raise questions about accessibility and fairness, especially for SCD patients in sub-Saharan Africa. Traditional treatments, while not curative, are significantly more affordable.

In conclusion, Casgevy and Lyfgenia are indeed milestones, but they are not the endpoint for SCD. The emergence of gene editing therapies marks the beginning of a new era, providing hope for revolutionary treatments. However, challenges such as safety concerns, accessibility, and pricing highlight the need for continuous advancements and ethical considerations in the evolving landscape of gene therapies. As we witness these groundbreaking treatments, Winston Churchill’s words come to mind: “Now this is not the end. It is not even the beginning of the end. But it is, perhaps, the end of the beginning.” The advent of these therapies signifies a new chapter, and while celebrating their potential, we must also address the challenges and questions they bring.

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The Anhui Provincial Peptide Drug Engineering Laboratory (University of Science and Technology of China), in collaboration with a team from Nanjing University, has reported the latest advances in the field of asymmetric radical acylation achieved through photoenzyme catalysis in the journal "Nature."

In recent times, the team led by Professor Tian Changlin from the Anhui Provincial Peptide Drug Engineering Laboratory (Biomedical Department, University of Science and Technology of China, and the High Magnetic Field Science Center, Chinese Academy of Sciences) collaborated with Professor Huang Xiaoqiang's team and Professor Liang Yong's team from Nanjing University to make significant strides in the field of photoenzyme catalysis.

In response to the developed dual catalytic system involving thiamine diphosphate (ThDP)-dependent enzymes and photocatalysis using phosphorus-amino acid (ThDP) as the catalyst, various reaction intermediates, such as free radicals in many reaction processes, changes in the oxidation state of metal catalysts involved in the catalytic reaction, and electron transfer processes during oxidation-reduction, were identified and analyzed using electron paramagnetic resonance (EPR) methods. Professor Tian Changlin's team at the School of Life Sciences, University of Science and Technology of China, has long been engaged in research at the High Magnetic Field Science Center of the Chinese Academy of Sciences, focusing on the identification of free radicals and analysis of electron transfer in research related to high-field EPR equipment setup, low-temperature EPR method development, and the mechanisms of chemical catalysis and enzyme catalysis, achieving a series of research results (Nat Catalysis 2023; Angew Chem Int Ed 2023, PNAS, 2023, 2022; ACS Catalysis 2023, 2021; Chem Commun, 2022, 2021; Science 2018, etc.). Recently, Professor Tian Changlin's team collaborated with Professor Huang Xiaoqiang's team and Professor Liang Yong's team at Nanjing University to make significant progress in the field of photoenzyme catalysis. Using EPR methods, they identified the free radical intermediates in the newly developed catalytic system and the electron transfer mechanism in the catalytic reaction. The research results, titled "A light-driven enzymatic enantioselective radical acylation," were published in Nature (DOI: 10.1038/s41586-023-06822-x).

Biomanufacturing is one of the most promising green technologies for transforming industrial sustainability and is a core aspect of enzyme catalysis in synthetic biology. The combination of enzyme catalysis and photocatalysis, known as photoenzyme catalysis, integrates the diverse reactivity of photochemistry with the high selectivity of enzymes, making it the forefront strategy for developing new enzyme functions. The collaborative research team, using a combination of biomimetic and chemical simulation approaches (Figure 1), harnessed visible light excitation and directed evolution to extend enzyme catalytic functions to radical-radical cross-coupling. Additionally, by using directed evolution to modify ThDP-dependent enzymes, they reshaped ThDP-dependent benzaldehyde lyase into a radical acyl transferase (RAT), achieving a non-natural high enantioselective radical-radical coupling reaction.

The collaborative team explored the catalytic system of organic dye Rose Bengal and ThDP-dependent enzyme using 4-methoxybenzaldehyde 1a and free radical precursor oxidation-reduction active ester 2a as template substrates. Subsequently, a small and refined mutant library was constructed through molecular dynamics simulations and semi-rational design. The optimal mutant enzyme with high substrate tolerance and substrate selectivity (enantioselectivity up to 97% ee) was obtained, highlighting the finely tuned role of the enzyme's adjustable active pocket in the stereochemical control of free radical stereochemistry (Figure 2).

For the photoenzyme dual catalytic system, Professor Tian Changlin's team applied low-temperature (80K) electron paramagnetic resonance (EPR) experiments, capturing the ThDP-derived ketyl free radical (Int. B). Through EPR spin trapping experiments, they detected characteristic six-line splitting spectra in the standard reaction system, confirming it as an intermediate benzylic radical (Int. C) and the free radical product after addition with the capture agent. This provided direct evidence for unraveling the key to the new enzyme reactivity and the source of high stereochemical selectivity.

The collaborative development of a dual catalytic system combining ThDP-dependent enzyme catalysis and organic photosensitizer Eosin Y catalysis, led by Nanjing University and involving the team from the University of Science and Technology of China, not only transformed natural benzaldehyde lyase into a light-driven radical acyl transferase but also achieved excellent stereochemical control of a challenging prochiral free radical. Nanjing University is the first and last corresponding author unit, and the University of Science and Technology of China and the Anhui Provincial Peptide Drug Engineering Laboratory are co-corresponding author units. The aforementioned research work received funding from the National Natural Science Foundation of China's Outstanding Youth Fund, major instrument development projects, and the Ministry of Science and Technology's key research and development program.

The handling of peptide ends, specifically the N-terminus (amino terminus) and C-terminus (carboxyl terminus), depends on the specific requirements of the experiment or application. The decision to keep the ends free or block them is influenced by factors such as stability, reactivity, and the intended use of the peptide. Here are considerations for both scenarios:

1. Free Peptide Ends:

• N-Terminal Free: Leaving the N-terminus free can be suitable when the peptide is used for specific applications, such as immunizations, where the free N-terminus might be necessary for antibody recognition.
• C-Terminal Free: The free C-terminus is often preferred in situations where the native carboxyl terminus is essential for biological activity or when the peptide is intended for further modifications.

2. Blocked Peptide Ends:

• N-Terminal Blocking: Blocking the N-terminus can be done by acetylating it (adding an acetyl group). This acetylation can improve the stability of the peptide and prevent side reactions.
• C-Terminal Blocking: Blocking the C-terminus can involve amidation, adding a protecting group, or cyclization. Blocking the C-terminus can enhance stability and prevent unwanted reactions.

Considerations:

• Stability: Blocking the ends of peptides can enhance their stability against enzymatic degradation, particularly by exopeptidases.
• Reactivity: Unblocked ends may be more reactive and susceptible to side reactions. Blocking can reduce reactivity and increase the specificity of the peptide.
• Solubility: The solubility of peptides can be influenced by the presence or absence of terminal modifications. Blocking may affect solubility, and considerations should be made based on the peptide sequence.

Applications:

• Biological Assays: For peptides used in biological assays, blocking or leaving ends free may depend on the specific binding requirements. For example, if the peptide is mimicking a natural ligand, leaving the ends free might be preferable.
• Synthetic Strategies: In peptide synthesis, blocking can be important to control the direction of the synthesis and prevent unwanted reactions.

Summary:

• The decision to keep peptide ends free or block them depends on the specific requirements of the experiment, stability considerations, and the intended use of the peptide.
• Blocking can enhance stability, reduce reactivity, and prevent side reactions but may impact solubility.
• Considerations for leaving ends free include preserving natural functionality and recognizing specific biological interactions.

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Peptides and amides are related chemical compounds, but they have distinct differences:

Peptides:

1. Composition: Peptides are short chains of amino acids linked together by peptide bonds. They consist of amino acid residues joined by peptide bonds (C-N).
2. Function: Peptides serve various biological functions, acting as signaling molecules, hormones, enzymes, and structural components in living organisms.
3. Size: Peptides are typically smaller than proteins, generally consisting of fewer than 50 amino acids.
4. Classification: They can be classified based on their length as dipeptides (two amino acids), tripeptides (three amino acids), oligopeptides (several amino acids), or polypeptides (many amino acids).

Amides:

1. Chemical Structure: Amides are a class of organic compounds characterized by the functional group CONH₂. They contain a carbonyl group (C=O) bonded to a nitrogen atom.
2. Formation: Amides can be derived from carboxylic acids by replacing the -OH group with an amino group (-NH₂) or ammonia derivatives. The linkage in peptides is a specific type of amide bond called a peptide bond.
3. Diversity: Amides have a broader range of compounds beyond peptides, including simple amides like acetamide, complex pharmaceuticals, and polymers like nylon.
4. Uses: Amides have various applications, including pharmaceuticals, organic synthesis, materials science, and as solvents or plasticizers in industry.

In summary, peptides are a subset of compounds formed by amino acids linked through peptide bonds, while amides encompass a larger group of compounds that include peptides but also extend to other chemical structures with the CONH₂ functional group.

Peptide synthesis refers to the chemical process of creating peptides—short chains of amino acids linked together by peptide bonds. This process is crucial in various scientific, medical, and industrial applications due to the following functions:

1. Biomedical Research:

• Drug Development: Peptide synthesis helps in creating peptide-based drugs, including hormones, antibiotics, and antiviral agents. These synthesized peptides may mimic naturally occurring compounds or have modified structures for enhanced therapeutic effects.

• Biochemical Studies: Researchers use synthesized peptides to study biological processes, interactions, and signaling pathways within cells. This aids in understanding the functions of proteins and their roles in diseases.

• Vaccine Development: Synthesized peptides are utilized in the development of vaccines to trigger immune responses against specific pathogens or diseases.

2. Protein Engineering:

• Functional Analysis: Peptide synthesis allows the creation of peptides with specific sequences to investigate protein function, structure, and interactions.

• Designing Modified Proteins: By synthesizing peptides, scientists can modify or engineer proteins to improve their stability, activity, or binding affinity for various applications, including biotechnology and pharmaceuticals.

3. Diagnostic Tools:

• Biosensors and Probes: Synthesized peptides serve as probes or components of biosensors used for detecting specific molecules, analyzing biological samples, or diagnosing diseases.

• Diagnostic Testing: Peptides are employed in diagnostic tests, such as ELISA (Enzyme-Linked Immunosorbent Assay), for detecting biomarkers indicative of certain diseases or conditions.

4. Industrial Applications:

• Material Science: Peptide synthesis contributes to the development of biomaterials, including hydrogels and nanomaterials, for applications in tissue engineering, drug delivery systems, and regenerative medicine.

• Biocatalysis: Synthesized peptides can act as biocatalysts in various chemical reactions, contributing to the synthesis of other complex molecules.

5. Agricultural and Food Industries:

• Crop Protection: Peptide synthesis aids in the development of bioactive peptides used in plant protection and crop improvement against pests and diseases.

• Food Industry: Synthetic peptides find applications as additives, flavor enhancers, and preservatives in the food industry.

6. Peptide Libraries and Screening:

• High-Throughput Screening: Synthesized peptide libraries enable screening against specific targets, facilitating the discovery of compounds with desired biological activities, aiding drug discovery and development.

In essence, peptide synthesis plays a pivotal role across diverse fields, spanning from fundamental research to practical applications in medicine, biotechnology, diagnostics, materials science, and more.