Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration

Abstract

The use of artificial tissues in regenerative medicine is limited due to hypoxia. As a strategy to overcome this drawback, we have shown that photosynthetic biomaterials can produce and provide oxygen independently of blood perfusion by generating chimeric animal-plant tissues during dermal regeneration. In this work, we demonstrate the safety and efficacy of photosynthetic biomaterials in vivo after engraftment in a fully immunocompetent mouse skin defect model. Further, we show that it is also possible to genetically engineer such photosynthetic scaffolds to deliver other key molecules in addition to oxygen. As a proof-of-concept, biomaterials were loaded with gene modified microalgae expressing the angiogenic recombinant protein VEGF. Survival of the algae, growth factor delivery and regenerative potential were evaluated in vitro and in vivo. This work proposes the use of photosynthetic gene therapy in regenerative medicine and provides scientific evidence for the use of engineered microalgae as an alternative to deliver recombinant molecules for gene therapy.

Introduction

Oxygen is an essential molecule for cell metabolism. It plays a key role in tissue survival and regeneration, yet paradoxically, its appropriate delivery is one of the major problems for the clinical translation of non-vascularized tissue engineering approaches [1]. As a result, cells within bioartificial tissue constructs are stringently dependent on oxygen diffusion, which barely reaches few hundred micrometers and limits the construction of artificial tissues to non-clinically relevant sizes [2]. Besides the basic oxygen requirements, tissue regeneration relies on several bioactive molecules, such as growth factors, that control key processes including inflammation, angiogenesis and tissue remodeling [3], [4], [5]. Scaffold bioactivation with growth factors has been intensively investigated as strategy to enhance the regenerative potential of engineered tissues [6]. However, direct growth factor administration has major limitations related to the short biological half-life of the molecules, and the consequent need for repeated administration of large and potentially toxic bulk doses [7], [8]. Conversely, traditional gene therapy represents a better strategy to provide a constant growth factor supply. Yet, this approach raises ethical and technical concerns that affect their translation into clinical settings. For instance, the delivery of target genes often creates risk of oncogenicity, insertional mutagenesis and viral vector-related immune reactions [9], [10], while target tissue-specificity and high transduction efficiency are still unrealized for non-viral gene delivery methods [11], [12]. Additionally, sustained transgene expression is not guaranteed if the transgenic cell survival depends of local oxygen tension at the injection site.

Recently, we have introduced HULK (from the German Hyperoxie Unter Licht Konditionierung) as a novel concept to deliver oxygen into biomaterials independently of blood vessel perfusion. The main idea behind the HULK approach is that, by incorporating photosynthetic microalgae such as Chlamydomonas reinhardtii (C. reinhardtii) into artificial constructs, the local induction of photosynthesis could be able to supply the metabolic needs of bioengineered tissues in vitro [13] and in vivo [14]. To take HULK one step forward, in this work we evaluated the feasibility of implanting photosynthetic scaffolds in fully immunocompetent mice. Then, we explored the idea of using genetically modified microalgae to engineer photosynthetic scaffolds that, in addition to oxygen, could provide other pro-regenerative molecules to the wounded tissue. As a proof of concept, we created a gene modified C. reinhardtii strain that constitutively secretes the human vascular endothelial growth factor VEGF-165 (VEGF).

In this work we propose that the activation of biomaterials with gene modified microalgae could be used to locally deliver oxygen and other pro-regenerative molecules into bioartificial tissue constructs.