Improving on the FOXN1-TAT Fusion Protein Approach for Thymic Regeneration

FOXN1 is the master regulator of thymic growth and activity; the thymus is an unusually straightforward organ in this respect. One can make it grow and perform to a greater degree just by dialing up expression of this one gene in thymic tissue. Thymic regrowth is a desirable goal for the elderly, given that thymic atrophy occurs in everyone, and limits the production of T cells. It is a major contribution to immune aging.

One of the approaches that has been taken to achieve this goal of thymic regrowth is the delivery of a FOXN1 recombinant protein attached to the TAT domain derived from the HIV-1 virus, allowing it to enter the cell. Researchers published a study some years back in which intrathymic injection was used, an approach that is probably too risky to serve as a basis for human therapies. Without direct injection, one can't get enough of the protein into the thymus to be worth the effort.

Today's open access paper discusses an advance on that earlier work by the same team. The researchers further attach an additional binding domain to the FOXN1-TAT fusion protein to optimize uptake in thymic tissue, and inject the protein intravenously instead of directly into the thymus. This necessarily requires higher doses (and recombinant proteins remain expensive), but the team reports good results. This is an approach that could in principle be developed for human use, setting aside the more conservative concerns one might encounter regarding the use of TAT versus the adoption of other options for cell entry.

Recombinant FOXN1 fusion protein increases T cell generation in aged mice

The thymus is a specified immune organ that provides an inductive environment for the generation of T cells that play a critical role in the adaptive immune system. Although the thymus continues to export T cells throughout life, it undergoes a profound atrophy with age, a process termed thymic involution, resulting in decreased numbers and functional capacity of T cells in the older adult, which has direct etiological linkages with many diseases. Furthermore, T cell immune deficiency in the older adult is exacerbated when the immune system is insulted by chemotherapy, radiotherapy, infections (e.g. HIV), and preparative regimens for foreign tissue or organ transplants. Therefore, restoring thymus function in the older adult has important implications.

We have previously reported that intrathymic injection (i.t.) of a recombinant (r) protein containing FOXN1 and a protein transduction domain embedded in the HIV transactivator of transcription (TAT) protein increases the number of thymic epithelial cells (TECs) in mice that have undergone hematopoietic stem cell transplantation. Consequently, these mice had enhanced thymopoiesis, an improved thymic output and an increased number of naïve T cells in the periphery. However, i.t. injection may not be an ideal choice for clinical applications. 

It has been reported that chemokine CCL25 is highly expressed in thymic tissue, especially thymic stroma. CCR9 is the receptor for CCL25. Unlike other CC chemokine receptors, CCR9 shows a strict specificity for its ligand CCL25. It has been shown intramural injection of a fusion protein containing the N-terminal of CCR9 and IL-7 increased the content of IL-7 in the thymus as compared to injection of IL-7 alone.

In this study, we develop a rFOXN1 fusion protein that contains the N-terminal of CCR9, FOXN1, and TAT. We show here that, when injected intravenously (i.v.) into aged mice, the rFOXN1 fusion protein can migrate into the thymus and enhance T cell generation in the thymus, resulting in increased number of peripheral T cells. Our results suggest that the rFOXN1 fusion protein has the potential to be used in preventing and treating T cell immunodeficiency in the older adult. 


Would this compliment HSCT at LEF for RMR study?

Posted by: erasmus at March 1st, 2023 2:58 PM

Maybe this tech could help with the cost of producing recominant proteins?:

"One key element of the new system is that the researchers used a different type of cell in their bioreactors - a strain of yeast called Pichia pastoris. Yeast can begin producing proteins much faster than mammalian cells, and they can grow to higher population densities. Additionally, Pichia pastoris secretes only about 150 to 200 proteins of its own, compared to about 2,000 for Chinese hamster ovary (CHO) cells, which are often used for biopharmaceutical production. This makes the purification process for drugs produced by Pichia pastoris much simpler.

The researchers also greatly reduced the size of the manufacturing system, with the ultimate goal of making it portable. Their system consists of three connected modules: the bioreactor, where yeast produce the desired protein; a purification module, where the drug molecule is separated from other proteins using chromatography; and a module in which the protein drug is suspended in a buffer that preserves it until it reaches the patient.

Reconfiguring the system to produce a different drug requires simply giving the yeast the genetic sequence for the new protein and replacing certain modules for purification. With colleagues at Rensselaer Polytechnic Institute, the researchers also designed software that helps to come up with a new purification process for each drug they want to produce. Using this approach, they can come up with a new procedure and begin manufacturing a new drug within about three months. In contrast, developing a new industrial manufacturing process can take 18 to 24 months."

"The researchers also wanted to ensure that their vaccine could be manufactured easily and efficiently. Many protein subunit vaccines are manufactured using mammalian cells, which can be more difficult to work with. The MIT team designed the RBD protein so that it could be produced by the yeast Pichia pastoris, which is relatively easy to grow in an industrial bioreactor.

Each of the two vaccine components - the RBD protein fragment and the hepatitis B particle - can be produced separately in yeast. To each component, the researchers added a specialized peptide tag that binds with a tag found on the other component, allowing RBD fragments to be attached to the virus particles after each is produced."

Posted by: jimofoz at March 1st, 2023 3:26 PM

They could probably even go one better by using full AND logic to target the two cell surface receptors using CO-LOCKR:

"The tool they created is called Co-LOCKR, or Colocalization-dependant Latching Orthogonal Cage/Key pRoteins. It consists of multiple synthetic proteins that, when separated, do nothing. But when the pieces come together on the surface of a targeted cell, they change shape, activating a sort of molecular beacon.

The presence of these beacons on a cell surface can guide a predetermined biological activity - like cell killing - to a specific, targeted cell.

The team showed that Co-LOCKR can focus the cell-killing activity of CAR T cells. In the lab, they mixed Co-LOCKR proteins, CAR T cells, and a soup of potential target cells - some had just one marker, others had two or three. Only the cells with the predetermined marker combination were killed by the T cells. If a cell also had a predetermined "healthy marker", that cell would be spared."

Designed protein logic to target cells with precise combinations of surface antigens (2021)

"Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells based on specific combinations of proteins present on their surfaces. We design colocalization-dependent protein switches (Co-LOCKR) that perform 'AND', 'OR', and 'NOT' Boolean logic operations. These switches activate through a conformational change only when all conditions are met, generating rapid, transcription-independent responses at single-cell resolution within complex cell populations. We implement 'AND' gates to redirect T cell specificity against tumor cells expressing two surface antigens while avoiding off-target recognition of single-antigen cells, and 3-input switches that add 'NOT' or 'OR' logic to avoid or include cells expressing a third antigen. Thus, de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single output."

Posted by: jimofoz at March 2nd, 2023 4:14 PM
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