Healthcare investing company Syncona has committed £85m to Freeline in an £88.4m Series B financing, it announced on Tuesday.
The FTSE 250 firm described Freeline as a "leading", clinical stage AAV gene therapy company focused on liver expression for chronic systemic diseases.
Syncona founded Freeline with Professor Amit Nathwani from University College London in 2015.
It said the Series B financing would enable it to drive its lead programme in Haemophilia B through the clinic, further broaden its pipeline, build on its core manufacturing capabilities and ultimately deliver its products to patients.
Syncona said it remained the sole institutional investor in Freeline alongside UCL Technology Fund, which committed £3.4m to the Series B round.
It added that it now had an 80% fully diluted stake in Freeline, which was valued at cost and held at £63.5m following the first tranche of the Series B investment.
"In line with our strategy of building global leaders, we believe in backing our companies strongly over the long term," said Freeline chairman and Syncona chief investment officer Chris Hollowood.
"We are demonstrating this approach with our substantial ongoing commitment to Freeline, a company we founded in 2015 and which has made significant positive progress."
Hollowood said the business had a "world leading" founder and executive team with extensive track records in gene therapy, clinical translation, early and late stage development and global drug approvals.
"With its first programme now in the clinic we look forward to continuing to work with Freeline as it seeks to progress its treatments to patients."
Anne Prener, chief executive of Freeline, added that the company was "delighted" to have secured Series B financing with Syncona and UCLB.
The proceeds, Prener explained, would enable Freeline to drive the business forward "ambitiously" in its next stage of growth and development.
"Freeline is a promising company at the forefront of an exciting area, with an industry leading, fully integrated AAV gene therapy platform.
"We have a deep pipeline of programmes and proprietary manufacturing capability enabling commercial quality and scale which we look forward to building out further in coming years."