CRISPR-edited hyper-secreting cells improve wound healing in diabetic mice

CRISPR hypersecreting cells for diabetics

Date: 21st May 2020

Cell therapy technology is rapidly evolving and whilst we now have several approved cell therapies for clinical use, their applications still remain somewhat limited despite their vast promise.  Now, scientists have engineered a Cas9-AAV6-based genome editing tool platform in which they have edited mesenchymal stromal cells to generate hyper-secreting cells that can improve wound healing in diabetic mouse models.

One of the first questions during cell therapy generation is to decide which cell type to use – ideally one that can be easily manufactured, and is safe to use. Mesenchymal stromal cells (MSCs) fit both criteria and have been used in particular in the treatment of inflammatory diseases and tissue injury.  As these cells do not exhibit long-term engraftment, they also offer short term benefits without the risk of integrating into the body.

However, whilst these cells have been exploited for their intrinsic anti-inflammatory actions, there has been important biological and pharmacological disparities in pre-clinical research and human translational studies. It is thought that at least some of these translational inconsistencies could be resolved by genetic engineering for example enhancing the anti-inflammatory, immunomodulatory or angiogenic effects of the MSCs and thereby strengthening their therapeutic potential.  However, the ability to genetically manipulate or engineer human MSCs for preclinical and clinical studies up until now has remained limited, especially with respect to gene integration, here precise genetic engineering is the ultimate goal.

Now scientists from Stanford University School of Medicine, US, led by Matthew Porteus have re-purposed a Cas9-AAV6 genome-editing platform for use in primary human MSCs.  They have generated a site-specific safe harbour gene integration system, and characterised the therapeutic efficacy of Cas9-AAV6-engineered hMSCs as a putative treatment of impaired wound healing.

Chronic wound healing is often compromised in diseases such as diabetes; an area in which MSCs could be of great potential therapeutic benefit.  With a current lack of multi-factor treatments for diabetic-induced ulcers, and only a 50% success rate of the current and only FDA-approved topical gel treatment – recombinant platelet-derived growth factor beta (PDGF-BB), this is an area that MSCs could make a real difference.

CRISPR-Cas9 AAV platform

The team began by tailoring their previous Cas9-AAV6 genome-editing platform to MSCs, showing that effective gene integration could be achieved in human MSCs derived from bone marrow (BM), adipose tissue (AD), and umbilical cord blood (UCB).  To achieve this, the CRISPR-Cas9 components of the system (Cas9 and single guide RNAs) were delivered into MSCs by electroporation either as mRNA or as a ribonucleoprotein (RNP) complex, and the adeno-associated virus (AAV) delivered the homologous repair template via transduction, to achieve transgene integration.

Delivery of a reporter gene was used to assess the system initially and to determine which of 3 loci would be the ideal ‘safe’ harbour.  After, successfully delivering the reporter transgene into MSCs and demonstrating that reporter protein expression peaked at 4 days post targeting (with expression sustained through at least three passages of cells), the team determined that the safe harbour of Hemoglobin Subunit Beta (HBB) was the most ideal, and that the gene manipulation did not alter the characteristics of the cells.

Bioactive cells

But would these cells survive and be bioactive?  To answer this the team generated dual-reporter-expressing MSCs and injected them into the wound bed of db/db mice which have impaired wound healing.  These mice have a leptin deficiency and are currently the most widely used mouse model of type 2 diabetes mellitus (T2DM). Following injection into these mice, the injected MSCs remained localised to the injection site, and the cell survival time ranged from 9-13 days with those cells located in non-wounded skin surviving for a greater period on average.

As this work showed initial promising survival rates, the team wanted to further test the system by engineering the cells to accelerate wound healing. Here they engineered cells to express growth factors and cytokines—PDGF-BB, VEGFA165, and IL-10 all; of which contribute to immune regulation and cellular migration and proliferation in wound healing.

By doing this, the team found that PDGFB and VEGF MSCs injected into wound beds had significantly smaller wound areas 7-9 days post injection compared with controls.  Furthermore, the time for the wounds to completely heal was faster in these mice. In contrast to this, IL-10 did not appear to enhance wound healing.

These two potential therapeutic MSC lines were found to address different aspects of wound healing. PDGFB MSCs showed improvements in granulation tissue formation whilst VEGF MSCs had enhanced blood vessel density.  Surprisingly perhaps, the combined injections of both MSCs together did not enhance wound healing above that of VEGF MSCs alone (which exhibited the most rapid healing).

Conclusion and future applications:

The team here were able to demonstrate integration of up to 3.2 kb of DNA as a stable transgene in hMSCs derived from multiple tissue sources.

In diabetic mouse models they have shown the therapeutic value of engineered MSCs to accelerate wound healing.  These effects are localised to the region of injection, but engineered MSCs could also be integrated with tissue engineering-based treatment such as hydrogel for similar results.

The ability to manipulate MSCs from different sources will be useful for a large number of clinical applications and valuable for many investigators in the extensive MSC research community. For therapeutic purposes, the system uses modular cassette designs to accommodate changes of cassette elements, such that it is easy to change the therapeutic agent for delivery.

It is thought that the engineered therapeutic protein-expressing MSCs will be of greatest value for short-term treatments of injury without replacing the recipient tissue.  The advantage is that potential oncogenic effects – that are an inherent possibility with long-term treatments – are alleviated here due to the limited longevity of the injected cells.

It is hoped that this Cas9-AAV6-based genome editing tool will be an accelerating platform for engineering MSCs and enhancing their use in clinical applications.  By ‘super-charging’ the cells to express high levels of secreted factors it may help to create more consistent clinical outcomes.

It is estimated that In the United States alone, every year approximately 73,000 amputations of lower limbs (unrelated to trauma) are performed on people with diabetes.  Most of which stem from ulcers that will not heal.  The potential of wound-healing therapeutic cells to change the lives of thousands of diabetes sufferers worldwide holds much promise.

 

Srifa, W., N. Kosaric, A. Amorin, O. Jadi, Y. Park, S. Mantri, J. Camarena, G. C. Gurtner and M. Porteus (2020). “Cas9-AAV6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice.” Nature Communications 11(1): 2470.

https://doi.org/10.1038/s41467-020-16065-3