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Hypothesis

Purging Autologous HSCs via Detection of Clonal Mutations

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Submitted:

02 October 2024

Posted:

02 October 2024

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Abstract
Allogeneic hematopoietic stem cell (HSC) transplants are usually used in cases where patients have active cancerous illness in their bone marrow, but allogeneic transplants can cause graft vs. host disease. If autologous HSCs could be purged of cancer cells, they could then be used for transplantation purposes without concern. It is still controversial whether purging is necessary, but it certainly would alleviate concerns. A novel method of purging is discussed herein that does not require chemotherapy and is targeted for each patient. It could be applied either in vitro or in vivo.
Keywords: 
Subject: Medicine and Pharmacology  -   Hematology

Introduction

A non-genotoxic, targeted method of purging hematopoietic stem cells (HSCs) that can overcome resistance could allow for safer autologous HSC transplantation [1].
Allogeneic HSC transplant is possible, but it often leads to graft vs. host disease[2].
If the patient’s blood cancer cells in circulation, as well as the lymph nodes and bone marrow, are sequenced[3], clonal mutations can be identified - i.e., mutations that are contained in all of their cancer cells. These mutations can be detected with molecular switches, and thus the cells can be eliminated using the effector module of said switches[4,5,6].
This process can be done ex vivo, using transfection or transduction. It can also be done in vivo via mobilization of patient HSCs and dual adenoviral vector transduction[7,8].

Purging Strategy

First, cancer cells in the patient’s bloodstream, lymph nodes, and bone marrow would be sequenced to determine his or her clonal mutations. The vector that is delivered to patient HSCs ex vivo or in vivo would have very broad tropism. It would be programmed to express CRISPRa modules that upregulate the expression of clonally mutated genes. It would also encode switches that can recognize the clonally mutated mRNA sequences and respond via activation of a toxin module that eliminates the cell [4,5,6]. One example of such a switch is RADAR, a system developed recently that can even detect point mutations in mRNA[9,10].
As described previously, CRISPRa expression could be halted via detection of the clonally mutated transcript somewhere other than the mutation site, as this would prevent long-term activation of the gene in non-cancerous cells [6].

Blood Cancer Elimination Strategy

The vector would also base edit the cd45 gene of the patient’s harvested HSCs so that anti-CD45 chimeric antigen receptor (CAR) T-cells can be intravenously administered to eliminate the remainder of the patient’s blood cancer cells[11]. The CAR T-cells can be purged as well if necessary, and should also have a base edited cd45 gene themselves to avoid fratricide. For acute myeloid leukemia, instead of cd45, a combination of flt3, cd123, and kit epitope editing can be exploited[12]. The CAR T-cells could potentially be off-the-shelf, precluding tumor cell contamination issues[13,14]. They could be eliminated after treatment and re-administered later if necessary[15]. An immunotoxin targeting CD45 could be used instead of CAR T-cells[16]. It was demonstrated that the base edited cd45 gene product for the immunotoxin referenced here[17] was more biophysically optimal than the one generated earlier for CAR T-cell targeting[11]. In either case, an anti-CD45 immunotoxin could be used to select HSCs base edited ex vivo. However, with regard to in vivo administration, repeated injections may be required for immunotoxins, as opposed to autologous CAR T-cells, which can persist in circulation for long periods of time. Finally, healthy HSCs with an edited cd45 gene, i.e., after selection, would be infused intravenously or selected in vivo. With regard to the ex vivo approach, treated cells could possibly be expanded in vitro if necessary [8,18]. In the latter case, a mixture of unedited and edited HSCs may home to the bone marrow. However, the edited versions would be selected over time and amplify. The edited HSCs would eventually reconstitute all the immune cell compartments.

Conclusions

As opposed to other purging strategies, this one would likely prevent escape variant issues caused by the decreased expression of a small number of cell surface proteins, as the vector would have very broad tropism. Additionally, it may be more resilient with regard to resistance to the cytotoxic element of the purge process, as a potent toxin targeting a necessary cellular component such as a ribosome-inactivating protein can be activated in response to clonal mutation detection.
The aforementioned purging strategy could ensure that autologous HSCs can be used in the context of blood cancer treatments, avoiding the issue of graft vs. host disease.
This clonal mutation targeting strategy can be used in the context of solid tumors as well, using oncolytic vectors that require detection of said mutations for replication and hyper-virulence[4,5,6].

Funding

None.

Conflicts of interest

None.

References

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